The main facts concerning the alterations in muscle blood flow, both during

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1 23 J. Physiol. (1953) 12, EXPERIMENTS ON THE POST-CONTRACTION HYPER- AEMIA OF SKELETAL MUSCLE BY S. M. HILTON* From the Physiological Laboratory, University of Cambridge (Received 5 November 1952) The main facts concerning the alterations in muscle blood flow, both during and subsequent to contraction, were described by Gaskell (1877). Gaskell then believed that the enormous hyperaemia following even short periods of contraction was brought about by vasodilator nerve fibres, which were stimulated concomitantly with the motor fibres themselves. When he later discovered (188) that lactic acid, a product of muscular activity, produced a vasodilatation in perfusion experiments, he advanced the theory that locally produced metabolites bring about the hyperaemia by a direct action on the blood vessels. Subsequently, Bayliss (191) showed that carbonic acid also produced a vasodilatation, and these results were confirmed in later experiments. Thus, Barcroft & Kato were able to assert (1915), in their paper on the effects of functional activity in striated muscle and the submaxillary gland, that, as an explanation of the hyperaemia found in both cases, Gaskell's theory was now unquestioned. Gaskell himself, however, did not reject the possibility that vasodilator fibres might play some role in the response, though he considered the extent of their contribution to be still an open question (1916). The matter has been raised again recently by Folkow & Uvnis (1948), who found that the cholinergic dilator fibres to the cat's skeletal muscle played no part in the general blood-pressure responses of the animal. These fibres can be activated by hypothalamic stimulation (Eliasson, Folkow, Lindgren & Uvniis, 1951), as part of a patterned response suggestive of a preparation for muscular activity, but no other function has as yet been definitely ascribed to them. Keller, Loeser & Rein (193) found that a small vasodilatation occurred in the resting muscles of the opposite limb following contraction of the hind-limb muscles, while Anrep, Blalock & Samaan (1934) found that this also occurred in the resting muscles of the same limb. These were true reflex effects, and were abolished by section of the nerve to the active muscle, but they did not appear to contribute * Work carried out during tenure of a personal grant from the Medical Research Council. Present address: National Institute for Medical Research, Mill Hill, London, N.W.7.

2 POST-CONTRACTION HYPERAEMIA 231 to a significant extent to the hyperaemic response in the active muscle itself. Both groups of authors attributed this response, in the main, to locally acting chemical factors. The traditionally accepted chemical factors (2 lack, lactic acid and ph changes) have now been virtually excluded from playing any major part in the response: in addition, there is only scanty evidence in the literature of the actual production of vasodilator substances by a contracting muscle. Anrep & von Saalfield (1935) first demonstrated the dilator properties of the venous effluent from an active muscle: the substance (or substances) concerned appeared to be very stable in blood. Fleisch & Weger (1937a) emphasized that such substances cannot be demonstrated under normal conditions; the flow through the muscle must be impeded in some way. Grant (1938) obtained indirect evidence in a human subject for the release of vasodilator substances. If the limb muscles are made to contract while their blood flow is occluded, a normal hyperaemic response is seen when the flow is re-established. The question still arises how the vasodilator substances, liberated by the active muscle cells, would affect not only the arterioles but also the small arteries which are widely dilated. Even arteries outside the muscle dilate following muscular contraction (Schretzenmayr, 1933; Fleisch, 1935). These effects cannot all be adequately explained on the basis of a direct action of metabolites on the vessel walls. Experiments of Schretzenmayr gave the first indication that muscular contraction might elicit vasodilatation through an axon reflex. He measured the calibre of the femoral artery in the inguinal region, and showed that it increased in diameter following activity in the muscles of the hind-limb. This dilatation was obtained independently of the central nervous system, but was abolished when the artery was painted with phenol below the site of measurement. He concluded that the phenol had destroyed nerve fibres travelling along the artery wall, thus interrupting the axon reflex responsible for the dilatation. Fleisch confirmed and elaborated these findings. He showed that the axon reflex could be elicited by local arterial injection into the muscle of various substances. Acetic acid, various intermediary products of metabolism, histamine and acetylcholine were all effective, the latter being extremely potent. Though these alterations in calibre of such large vessels will not ordinarily produce a significant change in the rate of blood flow, they are probably a reflexion of what is also taking place further down the arterial tree, where the effects on flow will be much greater. However, no experiments have yet been performed to show whether a similar mechanism is in any way responsible for the dilatation of the smaller vessels. In this paper, results are described of experiments which were performed in order to test the possibility that nervous pathways are essential for the full development of the hyperaemia following muscular contraction.

3 232 S. M. HILTON METHODS All the experiments were performed on cats under chloralose anaesthesia (7-75 mg/kg). The venous outflow from the gastrocnemiue muscle was isolated according to the method of Verzar (1912), except that the saphenous vein was tied off and the femoral vein itself was prepared for cannulation. Two lengths of polyethylene tubing were then passed into the vein, one pointing peripherally and the other centrally; and the blood was subsequently allowed to pass from the former, through a specially constructed drop chamber (Hilton, 1952) and straight back into the animal through the central length of tubing. Thus, the blood was long-circuited through what was stil a closed system. This technique permits of several hours of continuous working. without requiring any adjustment. The rate of formation of drops was recorded with a Gaddum drop timer (Gaddum & Kwiatkowski, 1938). It is difficult to keep the muscle, which has been freed from all but its upper bony attachments, adequately moist and at the correct temperature, so, for most of the experiments, the gastrocnemius was not stripped up and separated from the deep muscles of the posterior compartment ofthe lower leg. This means that the two or three very fine veins passing between the gastrocnemius and the deeper muscles were left intact, but this introduces no errors into the experiments here to be described. Through the original skin incision on the anterior aspect of the thigh a Perspex cuff bearing two platinum electrodes was applied to the tibial branch of the sciatic nerve, the peroneal branch having been sectioned. When direct stimulation of the muscle was required, two needle electrodes, insulated except for a few mm at the tips, were pushed through the skin into the gastrocnemius. The stimuli were condenser discharges from a neon stimulator at a rate of 3-4 per sec, the strength being such as to give a maximal or just submaximal contraction of the muscle. When required, arterial injections were made through a cannula tied into a branch of the femoral artery. After the main preparatory operation, the animal was left for an hour, and then finally set up. Heparin (1 units/kg) was administered before any vessels were opened. RESULTS The hyperaemia following a 1 see tetanus The extent of the post-contraction hyperaemia depends primarily on the duration of the contraction and the number of motor units that have been activated (Anrep et al. 1934). The rate of development, extent and duration of the hyperaemia following the standard 1 sec tetanus is seen in Fig. 1. Since the flow through the muscle is usually considerably impeded during such a tetanus, it seemed important to assess the part that reactive hyperaemia might be playing in the production of the post-contraction effect. The responses to occlusion for 3 sec of the venous outflow, and of the arterial inflow, are shown in Fig. 2. Occlusion of the venous outflow was not followed by any hyperaemia, but arterial occlusion did give rise to a hyperaemic response of comparatively short duration. Whereas its initial peak may be as high, or even higher than that of the response to a like period of maximal contraction, it is much more transient and is, therefore, insufficient to contribute to any significant extent to the post-contraction effect. The same conclusion was reached by Gaskell (1916). During a short period of venous occlusion, the muscle may be continuing to fill with blood; but this cannot account for the

4 POST-CONTRACTION HYPERAEMIA 233 c 7 A B - o o 12 *,24 -LE LI48) 14 - K. 1on Ti ime 3 sec Fig. 1. Records of venous outflow (Gaddum drop timer) from gastrocnemius muscle, and of arterial blood pressure. At signals, stimulation (1 sec at 4/sec) of tibial nerve. A: cat, 3-2 kg; B: cat, 1-5 kg, recorded with a faster drum speed. A B C _E~~~~~~~~~~I ~~ CL o ~ ~ ~ ~ ~ ~ ~ l24ī I1! Time 3sec - (v) (a) (v) (a) Fig. 2. Cat, 2-2 kg. Records of venous outflow from gastrocnemius muscle, and of arterial blood pressure. Effect of 3 sec occlusion of the femoral vein (v) and artery (a). B and C were recorded with a faster drum speed.

5 234 S. M. HILTON lack of response, for, even if the venous occlusion is maintained for several minutes, the hyperaemic effect is still negligible. On the other hand, prolonged occlusion of the arterial inflow is followed by proportionately larger hyperaemic effects. The post-contraction hyperaemia is also not the result of the concomitant electrical stimulation of nerve fibres which have a vasodilator action (i.e. either sympathetic or of the posterior root system), since, in a curarized animal -E ~ S75 A B C 12 B. P Time[fl 3 sec- Fig. 3. Cat, 2-8 kg. Records of venous outflow from gastrocnemius muscle, and of arterial blood pressure. Effect of intravenous injection of 1 5 mg/kg D-tubocurarine chloride between A and B. At signals A and C, stimulation (1 sec at 4/sec) of tibial nerve. At signal B, direct stimulation of the muscle (1 sec at 4/sec). in which neuromuscular transmission has been abolished, stimulation of the tibial nerve with the standard stimulus produces, if anything, a small diminution of flow (Fig. 3). This result is conclusive, for there is no recorded case in which curare has blocked either post-ganglionic sympathetic fibres or antidromic vasodilatation. Contraction of the muscle, on the other hand (produced by direct stimulation) is still followed by a hyperaemia. This, however, is less pronounced than usual, even though the stimulus strength had been increased. This diminution may well have resulted from a reduction in the number of muscle fibres activated, or it may represent a real reduction in the hyperaemic response. Nevertheless, the experiment shows that the main hyperaemic effect is intimately associated with the actual process of contraction itself. Anrep et ac. (1934) performed similar experiments on the dog, and arrived at the same conclusion.

6 POST-CONTRACTION HYPERAEMIA 235 The effect of drugs on post-contraction hyperaemia Atropine. If cholinergic vasodilator fibres are involved in the post-contraction effect, one might expect it to be abolished by atropine. As seen in Fig. 4 A, the hyperaemia was neither abolished nor even reduced by atropine in all doses examined (.5-9- mg/kg). The only effect occasionally observed was a slight potentiation of the vasodilator response after the smallest dose used (-5 mg/kg). This result does not preclude the participation of cholinergic fibres, for there are examples in the literature of the failure of atropine to abolish vasodilator effects produced by the stimulation of cholinergic fibres. Perhaps the best known is the case of the submaxillary gland, in which stimulation of the chorda tympani, after atropinization, still causes a brisk increase in blood flow when secretion is no longer obtained. 7 A B C 12 a sec Fig. 4. Records of venous outflow from gastrocnemius muscle, and of arterial blood pressure from three cats. Effect of intravenous injections of atropine, phentolamine and mepyramine on post-contraction hyperaemia. A: cat, 2- kg, after 4-5 mg/kg atropine. B: cat, 2-3 kg, after 1-5 mg/kg phentolamine. C: cat, 2-2 kg, after 5 mg/kg mepyramine. Phentolamine. This powerful sympatbolytic drug (Regitine, Ciba), even when given in large doses that considerably reduce the arterial blood pressure, did not diminish the post-contraction hyperaemia. This is illustrated in Fig. 4 B; the experiment was from an animal in which the contraction of the muscle did not reduce the blood flow through it. Mepyramine. Histamine is a natural constituent of skeletal muscle, and is, according to Anrep & Barsoum (1935), released during muscular contraction. If the release of histamine were responsible for the post-contraction hyperaemia,

7 236 S. M. HILTON the latter should be abolished, or at least reduced, by antihistamine drugs. However, mepyramine, even in large doses (Fig. 4C), had no effect. Cocaine. This local anaesthetic regularly reduced or abolished the postcontraction hyperaemia. Arterial injections of cocaine hydrochloride were not so effective in this respect as were direct injections into the muscle. 1 ml. of a 1 % solution, injected in divided portions, causes a small, transient vasodilatation which wears off in a minute or so. At this time the motor fibres are not yet paralysed, and stimulation of the tibial nerve produces a powerful A B Jec Fig. 5. Cat, 2F krg. Records of venous outflow from gastrocnemius muscle, and of arterial blood pressure. Effect on post-contraction hyperaemia of 1Omg cocaine (1:1) injected between A and B directly into the muscle. At signals, stimulation (1 sec at 4/sec) of the tibial nerve. contraction of the muscle which has almost no hyperaemic effect. This is shown in Fig. 5. It can be seen that the resting level of blood flow is the same after cocaine as before, so that the blood vessels can be assumed to be in a normal state of tone. A further indication that the vessel walls are not paralysed is given by the fact that acetylcholine produces its usual vasodilator effect, while the constrictor action of adrenaline is not only retained, but even potentiated. Injections of procaine into the muscle were much less effective. This is in keeping with the findings of Fleisch & Weger (1938), who performed a corresponding experiment on the dog's hind-limb perfused with blood. They added procaine to the blood to make a final concentration of 1: 2 and found that, after 1 min, the post-contraction hyperaemia was often abolished. Botulinugm toxin. This toxin is known to exrert a specific paralytic action on

8 POST-CONTRACTION HYPERAEMIA cholinergic nerve-endings by inhibiting the release of acetylcholine without affecting conduction or interfering with muscular contraction (Guyton & MacDonald, 1947; Burgen, Dickens & Zatman, 1949; Ambache, 1949). In four experiments 1-2,jg were injected in divided portions into the gastroenemius muscle under pentobarbitone anaesthesia. The animals were allowed to recover, and 2 days later the muscle was paralysed in every case, there being complete neuromuscular block. A B C L B16rFL Time 3 sec _ Fig. 6. Records of venous outflow from gastrocnemius muscle, and of arterial blood pressure. Effect of botulinum toxin on post-contraction hyperaemia. A and B: cat, 3-3 kg, 2 days after local injection of toxin into muscle. A: at signal, direct stimulation (1 sec at 4/sec) of muscle. B: at signal, arterial injection of 1~s acetylcholine chloride. C: normal cat, 2-3 kg; at signal, just-threshold stimulation (1 sec at 4/sec) of tibial nerve. At this time, the contractions produced by direct stimulation of the muscle were no longer followed by a hyperaemic response (3Fig. 6 A). As a control, the response of a normal muscle to a just-threshold contraction is shown in Fig. 6C. The absence of a post-contraction hyperaemia is particularly striking, because in the botulinized muscle the vasodilator response to arterial injections of acetylcholine is found to be much increased (Fig. 6 B). This hypersensitivity of the vessels to acetylchollne is in itself suggestive that the effect of the toxrin has been to paralyse cholinergic vasodilator fibres, for it is known that hypersensitivity of the denervated tissue to its transmitter results not only from actual nerve section but also when the nerve supply is functionally blocked for some time by drug action (Emmelin & Muren, 1952).

9 238 S. M. HILTON The role of true and axon rejlexes in the production of the post-contraction hyperaemia The finding that both cocaine and botulinum toxin abolish the post-contraction hyperaemia suggests that nerve fibres, probably cholinergic, are involved in the mechanism whereby the vasodilatation is brought about. The following experiments show that a true central reflex mechanism, however, is not responsible for this post-contraction effect. This bears out earlier contentions of Anrep and Rein (Keller et al. 193), already referred to. Neither removal of the lumbar sympathetic chains, nor interruption of the afferent fibres from the gastrocnemius by section of the posterior roots L 6-S 2, altered the hyperaemic response to standard stimulation of the tibial nerve. In sympathetic denervation experiments, both lumbar sympathetic chains were exposed, and the post-contraction hyperaemia was recorded before, and a few minutes after, removal of the chains. This procedure was not very satisfactory, for, as a result of the preliminary operation, the muscle blood flow was very fast and the hyperaemia was therefore relatively small. In the experiments in which the posterior roots were sectioned, this difficulty did not occur, because exposure of the roots did not affect the blood flow, and section of the roots increased the flow for a short period only. However, even these experiments do not definitely exclude the possibility of the participation of a true reflex, for the afferent fibres concerned could enter the spinal cord through higher lumbar roots. In order, therefore, to sever all nervous connexioris between the muscle and the central nervous system, the following experiment was performed. All branches of the femoral artery not supplying the gastrocnemius muscle were ligated and divided, great care being taken that no branch was missed: the femoral vessel itself was ligated below the origin of the branch to the gastrocnemius. The vein was prepared in the usual way. The only possible nervous connexions between the muscle and the central nervous system are now through its motor nerve or by any fibres that may run along the femoral artery. If the tibial nerve is now divided central to the point at which it is to be stimulated, the muscle blood flow at first increases but returns to normal in 15-2 min. At this time, contraction of the muscle has just the same effect on its blood flow as before section of the nerve (Fig. 7 A, B). The femoral artery is then divided and flow is reestablished by cannulation of the cut ends of the vessel. The muscle is now completely isolated from the central nervous system, yet the post-contraction hyperaemia is quite unaffected (Fig. 7 C). The final group of experiments to be described show that, if the postcontraction dilatation is due to an axon reflex, this reflex does not utilize fibres of the posterior root system, nor does it involve those autonomic fibres whose cell stations are in the ganglia of the sympathetic chains.

10 POST-CONTRACTION HYPERAEMIA 239 In two cats, the last two lumbar and the first two sacral posterior root ganglia were removed aseptically under pentobarbitone anaesthesia. Fifteen days later, an apparently usual post-contraction hyperaemia was obtained on stimulation of the tibial nerve (Fig. 8A). :12 OD II 6 A R C- I o _, 14r 1 Oo L Ti me losec I Fig. 7. Cat, 1-5 kg. Records of venous outflow from gastrocnemius muscle, and of arterial blood pressure. Effect of acute denervation on post-contraction hyperaemia. At signals, stimulation (1 sec at 4/sec) of tibial nerve. Between A and B, section of tibial nerve. Between B and C, femoral artery divided and re-connected by cannulation. c 75.E CL U. 'o j 12, U B.P. Fig. 8. Records of venous outflow from gastrocnemius muscle, and of arterial blood pressure. At signals, stimulation (1 sec at 4/sec) of tibial nerve. A: cat, 2- kg, 15 days after removal ofposterior root ganglia,l6-2. B: cat, 1-5 kg, 15 days after bilateral lumbar sympathectomy.

11 24 S. M. HILTON In two cats, bilateral lumber sympathectomy was performed under aseptic conditions and, in two other animals, the first two sacral sympathetic ganglia were also removed on both sides; 15 days later, tibial nerve stimulation produced in every case an apparently normal post-contraction hyperaemia (Fig. 8B). DISCUSSION The general assumption that reactive hyperaemia in muscle is, at least in part, a different phenomenon from the post-contraction effect is supported by the present findings. A brief period of occlusion of the arterial inflow to a muscle gives rise to a hyperaemia, but this is much more transient than the dilatation resulting from a like period of maximal contraction. Folkow (1949) came to the conclusion that Bayliss's original contention (192) concerning the origin of reactive hyperaemia was substantially correct. Bayliss had maintained that the smooth muscle of blood vessels 'possessed a natural tone, as does smooth muscle in any other situation, and that, as seen for example in the intestine, this tone will be altered in xesponse to changes in the stretching force to which the muscle is subjected. Thus, occlusion of the arterial inflow will lead to a diminution of the tone of the vessel walls beyond the block and, when flow is re-established, an initial hyperaemia will result. This conclusion enables us to explain the differential effect observed on occlusion of the arterial inflow and of the venous outflow; only occlusion of the arterial inflow leads to diminution of the tone of the vessel wall and thus produces the hyperaemia observed when the flow is re-established. Occlusion of the venous outflow, on the other hand, leads to an increase in the stretching force, and subsequently an increase in vascular tone. Hence, the finding that venous occlusion is not followed by reactive hyperaemia is in agreement with Bayliss's original contention. With regard to the post-contraction hyperaemia, any hypothesis as to the mechanism by which it is produced must take account of the observations that it is abolished by cocaine, procaine and botulinum toxin without a reduction in the response of the muscle vessels to vasodilator substances. These findings are most readily explained on the assumption that a nervous mechanism is involved in the hyperaemic response. Moreover, the specificity of action of botulinum toxin would indicate that cholinergic fibres are participating in this response, even though it is not abolished by atropine. It has long been known that the cholinergic vasodilator fibres of the chorda tympani to the tongue and submaxillary gland are resistant to atropine, and recently Folkow, Haegar & Uvnais (1948) have shown that the vasodilator fibres to skeletal muscle in the cat, although they are almost certainly cholinergic, are very resistant to the action of this drug. The possibility that these cholinergic fibres produce the hyperaemia directly,

12 POST-CONTRACTION HYPERAEMIA 241 having been stimulated together with the motor fibres in the tibial nerve, can be excluded, since stimulation of the nerve after curare did not produce vasodilatation, whereas direct stimulation of the curarized muscle produced a contraction followed by the hyperaemic response. This shows that the response is closely associated with the process of contraction and that, if it is produced by a nervous mechanism, the stimulus must originate in the contracting muscle itself. Stretch receptors cannot be involved, because the hyperaemic response persisted after sensory nerve degeneration. It is most likely that the stimulus is a substance (or substances) produced by the contracting muscle. It has always been assumed that vasodilator metabolites are produced during contraction, but they have been thought to act directly on the vessel wall. The results obtained with cocaine, procaine and botulinum toxin make this supposition unlikely: this applies particularly to the experiments with botulinum toxin, because the vessels of the paralysed muscle in this case were found to be hypersensitive to the dilator action of acetylcholine. Whatever the nervous mechanism involved in the post-contraction hyperaemia, the fact that it is still obtained after all nervous connexions between the muscle and the central nervous system have been severed shows that it cannot be a true central reflex. This conclusion applies to the contractions obtained by stimulation of the motor nerve, but it is not denied that vasodilator nerve fibres may be stimulated centrally during muscular exercise in the intact animal and contribute to the vasodilatation associated with muscular contraction under physiological conditions. The vasodilator substance (or substances), therefore, seem to produce their effect through an axon reflex. It has not yet been possible to establish exactly the fibres concerned in this mechanism. Since the post-contraction hyperaemia persists after degeneration of the sensory fibres in the last two lumbar and the first two sacral posterior roots, it is unlikely that fibres of the posterior root system take part in this axon reflex: if they did, it would have to be assumed that sensory fibres from the gastrocnemius muscle enter the central nervous system through other roots, higher in the spinal cord. The abolition of the post-contraction hyperaemia after treatment with botulinum toxin is further evidence against the view that the pathway for the reflex is in fibres of the posterior root system; for this toxin acts specifically on cholinergic fibres, while sensory fibres are not cholinergic. There remain, therefore, as the most likely pathway for the axon reflex, the post-ganglionic fibres of the sympathetic outflow. Axon reflexes in these fibres have been described in the skin. Werne (1925) first demonstrated long pathway axon reflexes of this type in fish. In man, a short pathway axon reflex, a pilomotor response to faradic stimulation, was first described by Lewis & Marvin (1927) and, more recently, Coon & Rothman (194) have shown that PH. CXX. 16

13 242 S. M. HILTON acetylcholine, nicotine and other drugs with like pharmacological actions, when injected locally in high dilution, activate this axon reflex in both man and the cat. In their experiments on human subjects, a sudomotor response occurred simultaneously with the contraction of the pilomotor muscles. Wada, Arai, Takagaki & Nakagawa (1952) have confirmed the occurrence of this sudomotor response and have firmly established that it is the result of an axon reflex. The particular significance of these findings for the present study resides in the fact that chemical substances readily stimulate the endings of cholinergic and adrenergic nerve fibres that are ordinarily regarded as having a purely efferent, motor function. These findings in the skin recall the work of Fleisch (1935), who found that contraction of skeletal muscle excited an axon reflex, producing dilatation of the main artery to the hind-limb and, further, that this reflex could be elicited by chemical substances, all of which might be produced during muscular activity. The nervous pathway of this axon reflex was not ascertained; it is conceivable that the fibres responsible belong to the sympathetic system, and that the same mechanism produces not only dilatation of the femoral artery, but also the dilatation of the vessels within the active muscle. The conclusion that the axon reflex producing the vasodilatation in the muscle is mediated by the sympathetic system has, however, to be reconciled with the fact that it was not abolished after chronic lumbosacral sympathectomy. The intermediate sympathetic ganglia, being situated dorsally in the course of the rami communicantes, and in the ventral roots (Wrete, 1935; Boyd & Monro, 1949; Randall, Alexander, Hertzman, Cox & Henderson, 195), will survive the operation and may assume in adequate measure the function of the neurones that have degenerated. A similar suggestion has been made by Krogh, Harrop & Rehberg (1922) to explain local vascular responses in the web of the frog's hind-leg to mechanical and chemical stimuli. They postulated the existence, in close association with the blood vessels, of a network of nerve fibrils in which conduction can take place in any direction, which does not degenerate completely after sympathectomy, and which may regenerate i4ependep~tly. The loss of a proportion of the neurones responsible for the axon reflex is especially unlikely to manifest itself when measurements of blood flow are being made. There is also the possibility that ganglion cells occur in the periphery in the muscle itself. Most histologists have denied the existence of such cells, but the question has been raised again by the recent studies of Meyling (1949) and Gairns & Garven (1952). There remains the question of the nature of the chemical stimulus for the axon reflex. 2 lack can be excluded; for Krogh showed (1919) that the tissue 2 pressure does not fall, but in fact rises, in an active muscle. The changes in hydrogen-ion concentration that occur physiologically were shown

14 POST-CONTRACTION HYPERAEMIA 243 by Fleisch (1921) to be too small to account for the increase in blood flow through active tissues, while Krogh (1922) quotes evidence showing that a C2 tension which will produce an acidity far higher than that ever occurring normally has an insignificant effect on the vessels, especially the arteries, of the frog's tongue. Lactic acid, injected into the muscle arterially, has very little vasodilator effect (Keller et al. 193), and thepost-contractionhyperaemia is unaffected when lactic acid formation is prevented by iodoacetic acid (Rigler, 1932). More recently, Gollwitzer-Meier (195) has shown that the changes in ph of the venous blood from a muscle, during and subsequent to activity, bear no relationship to the associated hyperaemia. It is not likely that histamine plays an important part, because the postcontraction hyperaemia was unaffected by mepyramine. The effect of acetylcholine was not reduced by cocaine and was in fact increased after treatment with botulinum toxin: nevertheless, acetylcholine cannot be completely excluded, for Fleisch (1935) has shown that it is a potent stimulator of the axon reflex which dilates the femoral artery. Other substances that have already been considered but remain to be critically examined are ATP (Rigler, 1932; Fleisch & Weger, 1937 b), and potassium ions (Dawes, 1941): there may be some as yet unknown vasodilator substance which excites the axon reflex, and it is also possible that the post-contraction hyperaemia is due not to any single factor but to several acting synergistically. SUMMARY 1. The mechanism was analysed whereby the post-contraction hyperaemia in the gastrocnemius muscle following stimulation of the tibial nerve is produced. For this purpose, the outflow from the muscle was recorded continuously in heparinized cats, the venous blood being passed through a specially constructed drop-chamber and straight back into the animal. 2. Reactive hyperaemia following occlusion of the muscle artery was so short-lived that the post-contraction effect cannot be the result of occlusion of the vessels during muscular contraction. No hyperaemia followed sh rt risos of venous occlusion. The reactive hyperaemia following arterial n8 explained, as suggested by Bayliss, by the loss of tone in the vessels consequent on a reduction in the stretching force applied to them. 3. The post-contraction hyperaemia is not the result of the direct stimulation of the vasodilator fibres in the tibial nerve; for, in a curarized animal, nerve stimulation led to a slight vasoconstriction, whereas direct stimulation of the muscle produced a post-contraction hyperaemia. 4. The post-contraction hyperaemia was not affected by the intravenous administration of atropine, phentolamine or mepyramine. 5. The post-contraction hyperaemia was reduced or abolished by local' 16-2

15 244 S. M. HILTON injection into the muscle of cocaine, procaine and botulinum toxin. These injections did not reduce the responsiveness of the vessels to the dilator action of acetylcholine; treatment with botulinum toxin actually rendered the vessels hypersensitive. This suggests that a nervous mechanism is involved in the postcontraction hyperaemia. 6. Complete severance of all nervous connexions between the muscle and the central nervous system did not affect the post-contraction hyperaemia which is, therefore, not a true central reflex, but probably an axon reflex. 7. The post-contraction hyperaemia was not affected by chronic posterior root ganglionectomy. 8. Chronic bilateral lumbosacral sympathectomy had no apparent effect on the hyperaemia, but this does not exclude the participation of the sympathetic system on account of the presence of 'intermediate' ganglia. 9. It is suggested that the post-contraction hyperaemia is mediated as an axon reflex by fibres of the sympathetic outflow, probably cholinergic. The cell stations of these fibres could be either largely in the 'intermediate' ganglia, or possibly in the periphery, in the muscle itself. 1. The chemical stimuli which may initiate the axon reflex are discussed. I would like to thank Dr W. Feldberg for his asistance during the preparation of this paper. REFERENCES AMBACHE, N. (1949). The peripheral action of Cl. botulinum toxin. J. Physiol. 18, ANREP, G. V. & BAsoum, G. S. (1935). Appearance of histamine in the venous blood during muscular contraction. J. Physiol. 85, ANREP, G. V., BLALOCK, A. & SAmAN, A. (1934). The effect of muscular contraction on the blood flow in the skeletal muscle. Proc. Roy. Soc. B, 114, ANREP, G. V. & VON SAATIrELD, E. (1935). The blood flow through skeletal muscle in relation to its contraction. J. Physiol. 85, BARcuo'r, J. & KATO, T. (1915). Effects of functional activity in striated muscle and the submaxillary gland. Philos. Trans. 27, BAYLISS, W. M. (191). The action of carbon dioxide on blood vessels. J. Physiol. 26, 32-33P. BAYLISS, W. M. (192). On the local reactions of the arterial wall to changes of internal pressure. J. Phy8iol. 28, BOYD, J. D. & MONRO, P. A. G. (1949). Partial retention of autonomic function after paravertebral sympathectomy. Lancet, ii, BuROEN, A. S. V., DIcKENS, F. & ZATmAN, L. J. (1949). The action of botulinum toxin on the neuromuscular junction. J. Physiol. 19, CooN, J. M. & ROTHmAN, S. (194). The nature of the pilomotor response to acetylcholine; some observations on the pharmacodynamics of the skin. J. Pharmacol. 68, DAWES, G. S. (1941). The vasodilator action of potassium. J. Physiol. 99, ELIASSON, S., FOLKOW, B., IaNDGREN, P. & UvNXs, B. (1951). Activation of sympathetic vasodilator nerves to the skeletal muscles in the cat by hypothalamic stimulation. Acta physiol. 8cand. 23, EMMEuN, N. & MUREN, A. (1952). The sensitivity of submaxillary glands to chemical agents studied in cats under various conditions over long periods. Acta physiol. scand. 26, FLEIScH, A. (1921). Die Wasserstofflonenkonzentration als peripher regulatorisches Agens der Blutversorgung. Z. allg. Phy8iol. 19, FLEsCH, A. (1935). Les r6flexes nutritifs ascendants producteurs de dilatation art6rielle. Arch. int. Physiol. 41,

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