(Received 5 April 1948) Although it is probable that functional derangements within the autonomic

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1 186 J. Physiol. (I949) Io8, I86-I96 6I2.0I7.4:6I2.I72.2 BRADYCARDIA OF CENTRAL ORIGIN PRODUCED BY INJECTIONS OF TETANUS TOXIN INTO THE VAGUS NERVE BY N. AMBACHE AND 0. C. J. LIPPOLD From The Department of Physiology, University College, London (Received 5 April 1948) Although it is probable that functional derangements within the autonomic nervous system may occur in tetanus (Courtois-Suffit & Giroux, 1918), they are usually overshadowed, in the general clinical picture, by the severe disturbances in the somatic nerves and their centres. Harvey (1939) has put forward the thesis that tetanus toxin produces a pathological change in the process of cholinergic transmission at motor nerve-endings, with apparent overactivity. If we accept his point of view there seems to be no reason why the vagus nerve should not exhibit a similar process resulting in bradyeardia. Clinically, however, there is no evidence for this; in fact the reverse.appears to be the case according to Dean (1917), who reported tachycardia (without pyrexia) in general tetanus. Experimentally this is also usually the case, unless the toxin is injected directly into the vagus nerve. This particular experiment was first performed by Meyer & Ransom (1903) on two dogs both of which developed bradycardia. The same phenomenon is obtainable in rabbits, and we have analysed this effect, which appears to be due to a central action of the toxin on *the vagal nucleus. METHODS The preparation and standardization of the CN 655 toxin, which was used for these experiments, has been described previously (Ambache, Morgan & Payling Wright, 1948). As before, the toxin was dissolved in sterile saline (0.9% NaCl), in the majority of experiments immediately before each injection. The experiments were all performed on rabbits of mixed stock and weighing between 0 45 and 3x2 kg. After the initial heart rate was measured at rest, the animals were anaesthetized with 26 mg./kg. body weight, of 'veterinary nembutal' (pentobarbital sodium) intravenously. The toxin, in doses ranging from 20 pg. to 1 mg., was injected with aseptic precautions either into the vagus nerve itself or in close approximation to it, in one of the following ways: (a) The vagus nerve was exposed in the neck and carefully separated from the common carotid artery. The nerve was raised gently on a sterile loop of silk thread at the level of the cricoid cartilage. A very fine short-bevelled syringe needle (no. 27 S.W.G.) was then inserted through the perineurium and enough toxin solution

2 VAGAL ACTION OF TETANUS TOXIN 187 ( c.c.) was injected in a peripheral direction to produce a small visible swelling within the nerve. The toxin was injected into one or other vagus nerve except in one animal in which it was injected into both vagi. In three rabbits which served as controls for (a) and (b) a mixture of toxin and antitoxin was injected centripetally into one vagus. The toxin for these controls was neutralized, just before the injections, with a 150-fold excess of antitoxin (Weilcome tetanus antitoxin globulins). (b) The vagus nerve was exposed on one side in the neck. It was firmly ligated and cut distal to the ligature; 2-3 in. of the peripheral end were avulsed. Toxin was then injected upwards into the ligated central end. An ascending flow of the toxin solution, which is yellowish, was clearly visible within the perineurium at the time of the injection and a small bubble of air from the dead-space of the syringe-needle was sometimes seen disappearing out of sight within the nerve. (c) The toxin was injected into the carotid sheath, away from the vagus and without touching the nerve. (d) The abdomen was opened by a midline incision and the stomach and oesophagus were delivered through the wound. After the vagus nerves were identified on either side of the oesophagus, the syringe needle was inserted through the anterio; peritoneal covering of the oesophagus. The toxin (mixed with indian ink in the first few experiments) was injected subperitoneally (i.e. between the peritoneum and the outer muscular layer of the oesophagus) in an upward direction, in a volume of saline of 1-2 c.c. Contact with the vagi was carefully avoided throughout this procedure. The control animals received either (i) similar amounts of toxin boiled for 2 min. or (ii) saline, or (iii) 1 mg. of toxin neutralized immediately before the injection with a 150-fold excess of antitoxin. (e) The stomach was delivered as in (d), but the toxin (mixed with indian ink in some of the experiments) was injected subperitoneally at 3-4 loci along the lesser curvature and on the anterior wall of the stomach. The injection (volume 1-2 c.c.) raised a small swelling between the peritoneum and Again, contact with the branches of the anterior vagus the outer muscular layer of the stomach. was avoided. After the animals recovered from the operation, they were examined at least once a day until their death. The apex beat was counted by auscultation through a stethoscope; several (from 5-10) consecutive 5 sec. counts were recorded. The mean of these was taken in each case as the heart rate per mi. The heart rate was recorded electrically in some of these animals, with the help of a direct writing 'Cardiotron' electrocardiograph for the loan of which we are indebted to Messrs Phillips andco. As the input to this instrument is balancedsymmetricallywith respect to earth, interference from the a.c. mains, and the need to screen the animal are eliminated. A chest lead was found in trial experiments to be the most satisfactory. Records taken with other leads (I and II) showed considerable attenuation of the cardiac potentials. Before the operatioxi the fur was removed, by shaving, from an area about 3-4 cm. in diameter on (a) the left anterior, (b) the right posterior chest wall and (c) on one thigh. At the time when records were taken an abrasive electrode jelly was rubbed into these areas and the three leads were either clipped on to the skin or strapped on tightly. The thigh lead was earthed and the two chest leads were connected to the input stage. E.c.g.'s were taken before, and at various intervals after, the injection of toxin. RESULTS Injections of toxin into the cervical vagus The injection of toxin into an otherwise intact vagus nerve, or into the central end of a cut vagus, on either side, brings about, after a latent period of hr., a considerable slowing of the heart (see Table 1). In the eight experiments listed in Table 1A, the mean maximal fall in heart rate was 176 beats/min. from an initial mean rate of 292/min. The onset of this bradycardia may be quite rapid; e.g. in Exp. 6 the heart rate dropped by 172 beats/min. in the 5 hr. between the two measurements taken at 17 and 22 hr.

3 188 N. AMBACHE AND 0. C. J. LIPPOLD TABLE 1. Bradyeardia in rabbits after injections of tetanus toxin into the vagus nerve in the neck The heart rates recorded in this and subsequent tables were measured initially before anaesthesia and injection, and at various intervals specified in parentheses, after injection. Dose of Resting heart rate (beats/min.) Rabbit Wt. toxin Site and direction A no. (kg.) (mg.) of injection Initial After injection (hr.) Remarks A. Active toxin x15 Rt. uncut; (23); 104 (41); peripheral 190 (55); 132 (63) *2 Lt. cut; (43); 90 (45) central end Lt. cut; central end (24) 4 1X6 05 Lt. uncut; peripheral (23) Lt. cut; central end (22); 94 (27) Lt. cut; (23); 108 (23.5) - ganglion nodosum Lt. cut; central end (17); 100 (22) See Fig Rt. cut; central end (20); 127 (46); 88 (47) B. Controls: toxin neutralized by an excess of antitoxin 9 1x Rt. uncut; (96) Died of uncentripetal known cause on 7th day Rt. uncut; (72); 345 (96); Survived centripetal 309 (235) 18 days Rt. uncut; (96); 344 (235) Survived centripetal 18 days C. Other controls (active toxin) Subperi (48) Injection-sit e ( + ink) cardial controlled iat autopsy (72 hr.) indian ink found between parietal pericardium and ventricle Subperi (48) cardial Carotid sinus (70) Severe region torticollis Intravenous (120); 330 (241) No symptoms after 10' days Intravenous (120); 285 (241) No symptoms after 10 days

4 VAGAL ACTION OF TETANUS TOXIN 189 Cardiac arrhythmia was always associated with the slowing. In the final stages of the condition complete ventricular stoppage occurred for brief periods (3-4 sec.) when the rabbits would show signs of distress and start struggling. The animal's condition may then worsen rapidly and unless active preventive measures are taken, death from heart failure occurs on the second or third day. A few experiments, not listed in Table 1, with doses of toxin less than 0x15 mg. also resulted in a fatal issue. Owing to a slight, inevitable, leakage of toxin out of the injected nerve into the surrounding muscles of the neck there was, in nearly all these experiments, a progressive torticollis towards the injected side. But this does not appear to be the initiating cause of the bradycardia because, in other experiments in which severe torticollis followed the injection of 0x1 mg. of toxin into the carotid sheath and of mg. into the carotid sinus region there was no bradycardia after 34 days, and, conversely, bradycardia without torticollis was produced by the abdominal types of injections (q.v.). In addition to these symptoms, the peripheral injections into the uncut vagus produced a laryngeal stridor which was sometimes related-to the inspiratory, sometimes to the expiratory, phase of respiration. This stridor was audible without the aid of a stethoscope but stethoscopic auscultation revealed that it was loudest over the larynx. It appeared to be due to tetanic spasms of various muscles in the larynx, but no detailed analysis of this effect was made because in the majority of subsequent experiments central-end injections were performed. The results of a few injections into the cut peripheral end of the vagus are at present equivocal and will not be considered here. Symptoms of general tetanus were not seen in any of these experiments. SpecdiJcity of the reaction. That these effects are specifically produced by the toxin is shown in the control experiments in Table 1 B, in which 0 5 mg. of toxin, neutralized immediately before the injection with an excess of tetanus antitoxin, was injected into the intact vagus on one side. There was no slowing of the heart and two of the animals survived for 18 days; the other died of an unknown cause on the seventh day, but without bradycardia. The injection of larger doses of toxin (0.9 and 1 mg.) mixed with indian ink into the heart itself, between the perietal pericardium and the ventricular muscle failed to produce bradycardia after 48 hr., when the animals were killed for autopsy. Two rabbits which received 0-17 and 0-28 mg. of toxin/kg. intravenously survived for 10 days without symptoms of tetanus or any alteration in heart rate (Table 1 C). Paravagal injections in the abdomen Perioesophageal injections. Cardiac slowing and irregularity was produced regularly by this type of injection (Table 2) with doses of toxin ranging from 0 3 to 1 mg. but the latent period before its onset was somewhat longer than with the injections into the cervical vagus. Again, and in contrast with the

5 190 N. AMBACHE AND 0. C. J. LIPPOLD first type of injection, it must be stressed that great care was taken to avoid any mechanical injury to the vagi by contact with the needle. The effect was produced by leaving the uninjured vagi bathed in the solution of toxin (mixed with indian ink in 1 experiment). Five of the animals died of heart failure; the rest were used for experiments. There were no symptoms of generalized tetanus in any of these animals. Injections of boiled toxin, saline, or of 1 mg. of toxin neutralized, just before the injection, with an excess of tetanus antitoxin, failed to produce either bradyeardia or death (Table 2B). TABLE 2. Bradyeardia after paravagal injections of tetanus toxin, between the oesophagus and its peritoneal covering in the abdomen Dose of Resting heart rate (beats/min.) Rabbit Wt. toxin nitial After injection(hr.)al no. (kg.) (mg.) Initial After injection (hr.) Remarks A. Active toxin (21); 108* (45) 265 (21); 276 (46); 72* (74) 3 0* (50); 234 (93); 209 (120) 310 (26) * *5 0* (25); 138t (46) 284 (48); 272 (76); 127 (96) 240 (45); 218t (51) (+ink) t (17) t (42); 84* (46) (27); 202 (44); 134* (72) B. Controls (boiled) (72); 288 (144) (boiled) (48) Saline (+ink) l mg.+ excess antitoxin 5 0*7 l mg.+ excess antitoxin 6 1*6 1 mg.+ excess antitoxin (72) (17 days); 435 (20 days); 390 (31 days) (14 days) (17 days); 258 (20 days); 306 (31 days) * Rhythm irregular. t Very irregular. Died hr. Died overnight (30-46 hr.) Died 48 hr. Died overnight (51-67 hr.) Died at 18-5 hr. Killed on 6th day; no slowing Killed 48 hr.; no slowing Killed 72 hr.; no slowing Survived 31 days Died of uinknown cause 17 days later Survived 31 days Anerior wall of the stomach. Bradyeardia and death followed the injections of toxin by this route (see Table 3). The injections were performed, again, without mechanical contact with the branches of the left vagus. Symptoms of general tetanus were absent.

6 TABLE 3. VAGAL ACTION OF TETANUS TOXIN 191 Bradyeardia after subperitoneal injections of tetanus toxin along the lesser curvature and anterior wall of the stomach Dose of Resting heart rate (beats/min.) Rabbit Wt. toxin r A no. (kg-) (mg.) Initial After injection (hr.) Remarks 1-05 (+ink) (3); 248 (23); 96* (46) (+ink) (2); 109* (45); 147 (70) 3t (+ink) (24); 96* (27) Died 28-5 hr * (21) Died 36 hr. 5 1F4 1 (+ink) * (45) Died 46 hr. * Irregular. t This animal had both abdominal vagi cut aseptically 4 days before injecting toxin. Analysis of the effect Removal ofbradycardia by vagotomy. To facilitate the subsequent performance of vagotomy without anaesthesia, a very fine sterile silk thread was looped carefully round the intact vagi in the neck at the time of the operation for injection. The ends of this loop were knotted together and brought out through a small hole between the skin-sutures in the midline. The free end of the thread was twisted round a short piece of matchstick which was stuck down to the skin with collodion. Loops were prepared round both vagi, except in the experiments on central-end injections in which it was only necessary to prepare one loop round the remaining intact vagus on the side opposite to the injection. When the time came for vagotomy, the matchsticks were cut away from the skin and the loops were. pulled out briskly, slicing through the vagi, the whole procedure being quite painless to the animal, as had been shown previously by Pavlov (1910). The vagi were divided after the development of cardiac slowing and at a time when the death of the animal appeared imminent within the next few hours. In all cases, the vagotomy removed the bradycardia (Table 4), indicating that this had been vagal in origin. One of these animals was allowed TABLE 4 Details and time Initial (in hr. after heart administration of Heart rate (beats/min.) rate toxin) of vagotomy A Rabbit (beats/ or of other Immediately no. Type of toxin injection min.) procedures Before after A. Abolition by vagotomy of the bradycardia produced by vagal tetanus 1 Lt. (cut) central end 264 Rt. cut at 27 hr Lt. (cut) central end 294 Rt. cut at 29 hr Lt. uncut 396 Both cut at 23 hr Paravagal 429 Both cut at 100 hr (oesophageal) B. Temporary removal of bradycardia by local anaesthesia (5% procaine) of the vagi in the neck 5 Rt. (cut) central end 260 Procaine (1 c.c.) at hr. 6 Paravagal 429 Procaine (0.5 c.c.) (oesophageal) at 97 hr.

7 192 N. AMBACHE AND 0. C. J. LIPPOLD to survive after the vagotomy. Its heart rate immediately after vagotomy was 307/min. Twenty-six hours later it was 306/min. and after 43 hr. it was 270/min. and regular. It died 2 hr. later (45 hr. after vagotomy), with gross ulcerations in its stomach. Gastric erosions have been found regularly at autopsy after all 3 types of toxin injection. It is hoped to publish a study of their pathology elsewhere. Exps. 1 and 2 in Table 4 A deserve especial consideration. In them the toxin was injected upwards into the central end of one cut vagus. The bradyeardia was removed by cutting the remaining intact vagus on the opposite side, showing that the slowing was produced entirely by an overactivity, of central origin, of that vagus. Hence, the centripetal injection of tetanus toxin into the vagus nerve on one side, produces a reflex activation of the other vagus. Effect of blocking the vagus trunk with a local anaesthetic. Local anaesthesia of the remaining intact vagus (or vagi) with c.c. of a 5% solution of procaine, injected into the tissues surrounding the vagus in the neck, was, like vagotomy, effective in removing the bradycardia (Table 4 B), but this action was not permanent and the bradycardia returned as the anaesthesia wore off. Atropine. The cardiac slowing of vagal tetanus could be abolished by a peripheral block of the vagi with atropine. However, it is well known that atropine is inactivated fairly rapidly by the serum and tissues of rabbits (Bernheim & Bernheim, 1938). For that reason it could not be expected to have any longlasting effect on the bradycardia. It did, however, have a considerable immediate effect in raising the heart rate, as shown in all the experiments in Table 5 and in Fig. 1. In Exp. 1 it was necessary to repeat the dose of atropine (0.6 mg./kg.) at 4 hr. intervals because of the transitory nature of its action. TABLE 5. Temporary removal by atropine of bradycardia produced by tetanus toxin Heart rate (beat/min.) Dose of atropine and time of A Initial heart administration (hr. after the Before the Immediately Rabbit no. rate (beat/min.) injection of toxin) atropine after 1* mg./kg. at 67 hr mg./kg. at 71 hr mg./kg. at 75 hr mg./kg. at 45 hr mg./kg. at 74 hr * mg. at 25 hr * Toxin was injected into the cervical vagus in these 2 animals; in the others, paravagally in the abdomen (oesophageal type of injection). Nembutal. In the early experiments, an attempt was made to obtain a tracing of the heart rate by recording the carotid blood pressure under nembutal anaesthesia. This attempt was foiled because the bradycardia disappeared after the administration of nembutal intravenously (Table 6). For that reason, it was decided, subsequently, to record the heart rate electrically in the intact, unanaesthetized, animals. The effect of nembutal on the heart rate in normal A

8 VAGAL ACTION OF TETANUS TOXIN 193 A *B C D E, F :>, &j J '_ z> X / G 4ii,s,4-'_v4 4, v.a~~ 1 sec. Fig. 1. Bradyeardia produced by vagal injections of tetanus toxin (anteroposterior chest lead). A, Initial heart rate (280/min.) after a rest period of 30 min. B, 12 min. after A and 7 mi. after the induction of anaesthesia with 58 mg. nembutal i.v.i. (heart rate 274/min.). 0 7 mg. of tetanus toxin was then injected into the central end of the left vagus, 3 in. of the peripheral end being avulsed at the same time. Next day (leads accidentauy reversed): C and D, 22-5 hr. after the injection: heart rate now very slow (96/min. in C and 109/min. in D) and irregular. Large waves due to respiration but heart beats clearly discernible, superimposed on these. The changes in amplitude of the R and T waves (T is not inverted), and in'the duration of the RT interval are probably incidental to the bradycardia, since they disappear 4*5 sec. later in E (q.v.). 0 5 sec. after the end of D anaesthesia was induced with 58 mg. nembutal i.v.i. and E starting 4 sec. later, shows the immediate return to the animal's original heart rate (281/min.). F, 140 min. later; the effect of nembutal is wearing off (heart rate 140/min.). 1 mg. of atropine was then administered intravenously and G, started 10 sec. later (heart rate rose to 250/min.). I mv= 12'5 mm.

9 194 N. AMBACHE AND 0. C. J. LIPPOLD rabbits is negligible (Table 6 B), except in occasional animals which had a heart rate below the average in this series, which was 275 beats/min. On the other hand, in the animals with vagal tetanus, nembutal restored the heart rate to its initial value or higher. This action appears to be a central one since, in the dose in which it was used in these experiments (26 mg./kg.), nembutal was found not to abolish the effect of stimulation of the peripheral end of the vagus nerve. TABLE 6 Initial heart Heart rate (beat/min.) rate before Time of administration of A- Rabbit injection of nembutal in hr. after the Before the Immediately no. toxin (beat/min.) injection of toxin nembutal after A. Temporary removal by intravenous nembutal (approx. 26 mg./kg.) anaesthesia of the bradyeardia produced by tetanus toxin hr hr hr hr hr B. Controls: effect of nembutal (approx. 26 mg./kg.) on normal rabbits DISCUSSION The results show that vagal or paravagal injections of tetanus toxin produce cardiac slowing, after a latent period which is characteristic of tetanus intoxications. Analysis of the effect suggests that it is another example of 'local tetanus', in this case of the cardio-inhibitory centre. This is shown most clearly in those experiments in which the toxin was injected into the central end, i.e. on the afferent side, of one vagus. The cardiac slowing, in experiments of this kind, is due entirely to efferent over-activity of the opposite vagus. The repetitive activity which initiates this type of reflex tetanus could originate either in the sensory nerve fibres on the afferent side, or in the nerve centre itself. However, it is known that the injection of toxin into a pure sensory nerve, if sufficiently remote from the spinal cord, does not elicit reflex tetanus (Fletcher, 1903; Fr6hlich & Meyer, 1916). On the other hand, if, as in our experiments, the toxin is injected sufficiently close to the C.N.S., for example into the dorsal roots (Fletcher, 1903), 'tetanus dolorosus' is produced. Fletcher's analysis of this type of 'sensory' tetanus, suggests, again, that the toxin does not act so much on the sensory nerve fibres themselves (or their dorsal root ganglia), as on the nerve centres in the cord with which these fibres are connected. In fact, once 'tetanus dolorosus' had developed, the dorsal roots could be sectioned close to the spinal cord without altering the syndrome.

10 VAGAL ACTION OF TETANUS TOXIN 195 The centripetal injection of the vagus in the neck forces a certain amount of toxin into the spaces between the nerve fibres and under the perineurium. The solution of toxin can be seen travelling upwards, at the time of the injection, probably in these spaces. Possibly it is directed to the brain stem in this way; this would present no serious anatomical problem. It was, however, surprising to find, when the toxin was placed in the vicinity of the vagi on the anterior wall of the stomach and oesophagus, that the characteristic reflex bradycardia was produced. This appears to indicate some sort of transport of the toxin from those regions to the vagal centre in the medulla. A similar transport, restricted to a single nerve-centre in the spinal cord, from an injection of tetanus toxin into the 'peripheral field' of that centre, has already been reported by Acheson, Ratnoff & Schoenbach (1942). In view of the absence of symptoms of generalized tetanus, the transport does not appear to be blood-borne but possibly intraneural. We have no evidence as to whether this transport would take place within the axons themselves or in the endoneurial spaces. In either case it would appear to be 'against the current'. For the contents of nerve axons are believed to be under pressure from the nerve-cell body and therefore constantly moving in a centrifugal direction (Young, 1944). Similarly, Weiss, Wang, Taylor & Edds (1945) have described a resting flow, distally, of the fluid in the endoneurial spaces. These authors performed very careful experiments with radioactive materials, but their results were obtained mostly within a few hours of nembutal anaesthesia, when presumably the animals were relatively immobile. However, Vishnevsky (1928) showed that methylene blue injected into the sciatic nerve moves centripetally if the limb is actively exercised. Moreover, and this may have a bearing on the pathology of tetanus, Ulyanov (1929) has shown that carmine, injected into the muscles of the calf, is driven up the sciatic nerve in the thigh when the calf-muscles are stimulated electrically. SUMMARY 1. The injection of tetanus toxin into the central end of one vagus nerve produces, in rabbits, cardiac slowing and arrhythmia. This localized symptom appears after a latent period of hr. and terminates in death from heart failure within a few hours, unless the remaining intact vagus is out. 2. The same effect could be produced by injecting the toxin (a) into an uncut vagus nerve, or (b) subperitoneally, in the vicinity of the vagus, either in its oesophageal portion or on the anterior wall of the stomach. 3. The reaction is specific and could, in all three types of injection, be prevented by tetanus antitoxin. 4. The cardiac slowing is due to an overactivity of the vagus. It is removed by vagotomy or by local anaesthesia of the vagi in the neck. It can also be abolished for a time (a) by a peripheral block of the vagi with atropine, and (b) by depression of the vagus centre with nembutal in anaesthetic doses.

11 196 N. AMBACHE AND 0. C. J. LIPPOLD 5. The analysis of this effect shows that the bradycardia is probably due to a central action of tetanus toxin on the cardioinhibitory centre. We are indebted to Prof. G. Payling Wright for providing us with the toxin, and to Miss Jean Barrett for helping us with the experiments, which were subsidized by a grant from the Central Research Fund of London University. REFERENCES Acheson, G. H., Ratnoff, 0. D. & Schoenbach, E. B. (1942). J. exp. Med. 75, 465. Ambache, N., Morgan, R. S. & Payling Wright, G. (1948). J. Physiol. 1G7, 45. Bernheim, F. & Bernheim, M. L. C. (1938). J. Pharmacol. 64, 209. Courtois-Suffit, M. & Giroux, R. (1918). The Abnormal Forms of Tetanus. (Translated by Bruce and Golla.). London: University of London Press. Dean, H. R. (1917). Lancet, 1, 673. Fletcher, W. M. (1903). Brain, 26, 383. Frohlich, A. & Meyer, H. H. (1916). Arch. exp. Path. Pharmak. 79, 55. Harvey, A. M. (1939). J. Physiol. 98, 348. Meyer, H. & Ransom, F. (1903). Proc. Roy. Soc. B, 72, 26. Pavlov, I. P. (1910). The work of the Digestive Glands, p London: Chas. Griffin & Co., Ltd. Ulyanov, P. N. (1929). Z. ges. exp. Med. 64, 78. Vishnevsky, A. S. (1928). Z. ges. exp. Med. 61, 107. Weiss, P., Wang, H., Taylor, A. C. & Edds, M. V. (1945). Amer. J. Physiol. 143, 521. Young, J. Z. (1944). Nature, Lond., 153, 333.