THE ACTION OF THE VAGUS ON THE SPLEEN. BY TEIICHI MASUDA (MUKDEN). (From the Physiological Laboratory, Cambridge.) BULGAK'S paper(1) in 1877 on the innervation of the spleen may be considered to be the first systematic study of this subject. A few years later Roy(2) published the results of his plethysmographic studies, in which he obtained a series of remarkable tracings with his "oncometer." These investigations showed that the main nerve supply of the spleen is derived from the splanchnic nerve, stimulation of which causes a powerful contraction of this organ. Very little work has been done, however, on the action of the vagus,on the spleen. As is well known, most of the viscera are innervated by two different nerve systems, namely, the para-sympathetic and the sympathetic, with actions entirely antagonistic to one another but maintaining a balance of tone under any given circumstances. It is surprising therefore that in the case of the spleen considerable work has been done on the action of the sympathetic while that of the parasympathetic still remains obscure, and it was with the object of throwing more light on this obscurity that the present research was undertaken. During the carrying out of this investigation, which was begun in October, 1925, a paper was published by Skramlik and Duran-Cao(3 treating of the same problem and reporting new facts which will be referred to later in this paper. Method. All my experiments were made on cats, in which animal, as the late Prof. Langley pointed out to me, the spleen is very rich in contractile tissue. Under urethane and E.C. mixture narcosis the abdomen was opened, a part of the omentum severed, and the spleen, freed from stomach and pancreas, enclosed in a special spleen box made of ebonite with a celluloid cover. In opening the abdomen I did not cut in the nipple line, as is usually done(4), but made one incision in the middle line, a second cut at right angles being made in the left side of the abdomen after ligaturing the muscular vessels. In this way the compression or twisting of the splenic vessels was eliminated to a great extent. The blood-pressure in the carotid artery was recorded simultaneously with the splenic contractions.
290 T. MASUDA. Results. (1) Stimulaion of the peripheral end of the vagus. As early as 1868 Oehl(5) stated that the stimulation of the peripheral end of the vagul provokes the contraction of the spleen. He arrived at this result by direct inspection of the change in volume and colour of the organ caused by the stimulation, and it was confirmed by Roy(2) by the plethysmographic method. Being convinced that the spleen communicates with the general circulation by a very narrow path and consequently is very little affected by a change in the systemic bloodpressure, Roy ascribed this contraction to the direct action of the nerve upon the spleen. Bulgak(l), on the other hand, observed no contraction and suggested that Oehl's results were very likely due to the change in respiration caused by the section of both vagi. Later Schafer and Moore(6), using the plethysmographic method, came to the conclusion that the stimulation of the peripheral end of the vagus had no effect upon the spleen. According to them Roy's results might have been caused secondarily by contractions of the cesophagus and stomach which they occasionally observed. The question, therefore, still remains undecided. In all my experiments stimulation of the peripheral end of the vagus in the neck always caused a more or less obvious diminution in the spleen volume and on some occasions was very marked (Fig. 1). This diminution, however, is only brief and is followed by a series of intensified wave-movements, and in a great many cases, after the initial contraction is over, the volume is slightly increased. After several minutes, however, the organ usually returns to its original volume. Fig. 1. Stimulation of the peripheral end of the So far my results agree with those of Oeh 1 vagus. Upper curve: and Roy, but doubt is thrown on their inter- blood-presse. Middl& curve: spleen volume. pretation by closer examination. In the first Lower curve: base-line place, the diminution in spleen volume usually of blood-pressure. Time, occurs a few seconds after the beginning of the fall in blood-pressure. In the second place, the change in volume is, generally speaking though not always, proportional to the change in blood-pressure: if the fall of blood-pressure is large, the diminution
ACTION OF VAGUS ON SPLEEN. 291 in volume is also large. Moreover, in some cases in which the stimulation was continued for a longer period, the volume varied as the bloodpressure rose and fell (Fig. 2). This fact seems to show that the volume Fig. 2. Long-lasting stimulation of the peripheral end of the vagus. Parallelism between blood-pressure and spleen volume. Upper curve: blood-pressure. Middle curve: spleen volume. Lower curve: time and ba-se-line of blood-pressure. Time, 1 sec. change is rather of a passive nature. Accordling to Roy (2) the plethysmographic tracing of the spleen shows specific waves, each of nearly one minute's duration, andl the indlividlual heart beats dlo not appear in the tracing. From this and other data, such as the fact that compression or even clamping of the abdominal aorta has very little effect on the spleen volume, he assumed that the path of communication betwe'en the spleen and the general circulation, as already stated, is narrow and that the spleen volume is relatively small or, at least, not very readlily affected by the change in bloodl-pressure. He therefore regardledl the contraction of the spleen as being dlue to the dlirect effect of the vagus.. If it could be shown that the spleen volume is affected by the heart beat, IRoy 's argument would become much less cogent, and this has actually been done by Schafer and Moore, who found that fine vibrations on their tracings were obviously due to the heart beat. Although this could not be confirmed in my own tracings, I have reasons for believing that the spleen volume may be influienced, as mentioned above, by changes in blood-pressure. After an intravenous injection of pilocarpine (1 or 2 c.c. of.1 p.c.. PH. LXII. 19
292 T. MASUDA. solution) the blood-pressure shows a typical fall with a simultaneous decrease in the spleen volume. If the dose of pilocarpine be large, the blood-pressure remains at this lowered level for a considerable time, the spleen remaining contracted as long as the low pressure persists, a fact which seems to point to the passive nature of the diminution of volume. Under favourable conditions both the change in blood-pressure and spleen volume produced by vagus stimulation could be brought about by an appropriate dose of pilocarpine (Fig. 3). In this tracing the Fig. 3. Pilocarpine injection and its effect on the spleen. 10c.0. of -1 p.c. pilocarpine hydrochloride injected into femoral vein at X. Upper curve: blood-pressure. Middle curve: spleen volume. Lower curve: time and base-line of blood-pressure. Time, 1 sec. passive nature of the change is naturally not so obvious as in the case of the larger dose, but i'f the findings are correct in that case., why sthould they not also be correct in the case where the spleen curve shows a closer resemblance to that produced by vagus stimulation? In order to confirm the passive nature of this spleen contraction, two methods; were adopted. In the first place, an attempt was made to eliiate the effect of blood-pressure by the use of Roberts's cornpensator(7). As, according to Roy(2 and others, the saline solution, especially in large amount, may itself cause dilatation of the spleen, de-
ACTION OF VAGUS ON SPLEEN. 293 fibrinated blood was used instead of Ringer's solution, and the compensator was accordingly modified to economise the blood. By inserting a cannula into the abdominal aorta and connecting this with the compensator, the blood-pressure could be kept practically unchanged even under vagus stimulation and no contraction of the spleen could be observed (Fig. 4). Fig. i'i 11 4h Fig. 4. Effect of blood-pressure on spleen volume. Upper curve: blood-pressure. Middle curve: spleen volume. Lower curve: signal and base-line of blood-pressure. Time, 10 sees. (a) Stimulation of peripheral end of right vagus. (b) Blood-pressure compensator connected and the peripheral end of vagus stimulated. Three minutes' interval between (a) and (b). In the second place, the vagus was stimulated inside the thorax below the point at which the nerve sends out its cardiac branch. In no case could even the slightest contraction of the organ be seen, whereas it is quite obvious in the same animal if a fall of blood-pressure is produced by stimulating the vagus in the neck, in spite of the fact that the 19-2
294 T. MASUDA. nerve has been cut inside the thorax, thus preventing the transmission of any impulse to the spleen. Skramlik and Duran-Cao(3) obtained similarly negative results and also made other interesting observations. According to them, the stimulation of the peripheral end of the vagus inside the thorax has no effect on the spleen volume, and further, if the vagus and the splanchnic be stimulated simultaneously, the splanchnic alone takes effect, while if the spleen be previously made to contract by stimulating the splanchnic and the vagus be then stimulated before the splanchnic effect dies away, the spleen begins to dilate more quickly than it might otherwise do. From this they conclude that the vagus dilates the spleen only when this organ is previously contracted by the effect of its antagonist, the sympathetic. In this way they thought to have proved that there still exists a certain antagonism between the vagus and the sympathetic in their action on the spleen. As plethysmography has some disadvantages, such as the exposure of the organ for too long, compression of the vessels, etc., they did not employ this method but simply observed the change in volume and colour of the organ, as some other workers have done. If the contraction takes place, the dark red colour is turned to light red and the smooth surface becomes granulated, which latter phenomenon is denoted by them as "Inselbildung." For instance, they observed in one case that the spleen contracted within 3*5 sec. of stimulating the great splanchnic nerve; Inselbildung occurred after 17 secs. and lasted 70 secs., the spleen becoming normal again after 105 secs. In another case in which the splanchnic and the vagus were stimulated successively, the spleen began to contract after 3-4 secs.; Inselbildung occurred after 20 secs. and lasted only 30 secs., total recovery taking place after 58 secs. Thus the time of recovery was reduced to nearly one-half. I have tried to repeat this observation but in no case have I succeeded in confirming their result. The results of one of my experiments are given below. The whole course of the change in volume is given in Fig. 5. There is clearly no difference between these two cases, and in two or three other experiments I obtained the same negative results 1. At least two factors come into question in considering the cause of this discrepancy. In the first place, the methods used were different. It is true that the plethysmographic method has disadvantages, as S k ram li k and D uran - Ca o indicate, and these disadvantages may 1 After this research was completed, Dr Hoet, working in the Cambridge Pharmacological Laboratory on the same subject, told me that he had obtained similar results to mine.
ACTION OF VAGUS ON SPLEEN. 295 Feb. 26, 1926. Cat No. 24. Under urethane and E.C. mixture and artificial respiration the thorax was opened and the vagus and the large splanchnic nerve in the left side made ready for stimulation. The spleen was put into the spleen box. Blood-pressure recorded in the right carotid. Time (secs.) Stimulation 0 Splanchnicus maj. of left side 5 7 15 39 41 Stimulation off 105 175 295 After 2 minutes: 0 Splanchnicus maj. of left side 3 7 26 42 Stimulation off 52 Left vagus 58 87 Stimulation off 107 207 300 B.P. mm. Hg. 157 160 162 190 175 175 162 160 150 151 153 154 181 166 160 157 155 155 158 152 Remarks B.P. begins to rise Spleen begins to contract B.P. reaches maximum Max. contraction of spleen. Decrease in volume of about 2 c.c. Spleen volume shows little variation Volume begins to increase Volume increase 1-4 c.c. Still increasing but more slowly Recovery of volume almost complete B.P. begins to rise Spleen begins to contract B.P. reaches maximum Spleen contraction maximum. Decrease of volume 1-8 c.c. Spleen remains contracted Spleen begins to dilate Increase of volume 1-5 c.c. Still slowly increasing Recovery almost complete 0 I I a 5 511 11w1a6o 2 Vagus St/n14/atbO, Spahnchn:c Stima/aaon IIII 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 200 Fig. 5. Effect of splanchnic and vagus on spleen volume. ----- Splanchnic stimulation followed by vagus stimulation. Splanchnic stimulation alone. Abscissa: time in seconds. Ordinate: spleen volume in c.c. 0: initial volume of spleen. be considerable if great care is not exercised; quite often no definite results are obtained, especially if the animal happens to be weakly. Taking all these drawbacks into account, however, it is scarcely credible
296 T. MASUDA. that my plethysmograph is not sensitive enough to record a change so evident as to shorten the time of recovery to one-half. In the second place, cats were used in my experiments, while Skramlik and Duran-Cao always used dogs. I do not know to what extent this difference may affect the results obtained. Taking all this into consideration, and with some reserve, I would say that the stimulation of the peripheral end of the vagus, even when it follows the sympathetic impulse, has no effect upon the spleen whatever. (2) Stimulation of the central end of the vagus. Bulgak(l) classified the nerves going to the spleen into two groups, namely, the motor and the sensory nerves. In the case of the former, contraction of the organ is manifested only on peripheral stimulation, while in the case of the latter it is shown only when stimulation is applied centrally. In the case of the vagus nerve he observed no contraction on stimulating either the peripheral or the central end. He thus excluded the vagus entirely from the list of the splenic nerves. Roy(2), on the contrary, always observed contraction of the organ on stimulation of either end of the vagus. Thus he found that stimulation of the central end of the vagus on one side, the other side being severed for the sake of eliminating the cardiac effect, was always followed by more or less marked contraction of the organ. Comparing this result with that obtained by stimulating the sciatic nerve, he regarded these two as analogous and thought that in this case the vagus was acting merely as a sensory nerve. Repeated trials on my part to obtain this effect resulted to my astonishment not in contraction but invariably in a more or less marked dilatation of the organ. At the same time a gradual fall in blood-pressure was observed (Fig. 6). This fall is typical of the action of the depressor nerve and led me to suppose that this dilatation was also due to the same cause. And such has actually been shown to be the case by Bayliss' who, using rabbits in which the depressor is running as an independent nerve, observed this same dilatation of the spleen, the only difference being that there was no after-contraction such as I observed in the spleen of cats (see below). Here again the change in volume of the spleen occurs a few seconds after the fall in blood-pressure. When stimulation ceases, the blood-pressure returns gradually to its initial height, the spleen 1 This experiment is not described in any of Bayliss's writings, nor indeed else. where, but the tracing is given in Starling, Principle8 of Human Physiology, to illustrate another subject and was unknown to me when this work was being carried out. Prof. Starling informed me that the tracing was given to him personally by Bayliss.
ACTION OF VAGUS ON SPLEEN. 297 contracts simultaneously, and there follow further several intensified wave-movements. If the stimulation be strong or continued longer, say Fig. 6. Stimulation of central end of vagus. Upper curve: blood-pressure. Middle curve: spleen volume. Lower curve: time and base-line of blood-pressure. Time, 1 sec. more than one minute, the blood-pressure rises higher than its initial height when the stimulation is over and the spleen shows also strong after-contraction, so that it becomes for a short time far smaller in volume than it was before the stimulation. In any case the wave-movements following this are generally more obviously intensified than is the case after stimulation of the peripheral end. In comparing this with the effect of stimulation of the sympathetic a marked resemblance is evident, and very possibly this after-effect is due to the secondary action of the increased secretion of adrenaline. B ayiiss (8) obtained similar results also in the submaxillary gland. of cats. He cut out the sympathetic going to the gland on one side, leaving the chorda tympani intact, and stimulated the central end of the vagus of the other side. In this way he observed the typical fall in blood-pressure, increased outflow of blood from the gland,. and also increased secretion of saliva. Considering that this result might be due to the action of the depressor nerve, he made further experiments to verify it. In cats the depressor fibres are said to run in the left side both in the vagus and in the sympathetic, while in the right side the vagus only contains them. He therefore took out these nerves on both sides and stimulated them separately, and observed in this way that stimulation of the central end of the right vagus, left vagus and sympathetic provoked the fall of blood-pressure and the increase of both
298 T. MASUDA. blood outflow and salivary secretion, while stimulation of the right sympathetic produced no result but a slight rise in blood-pressure. These responses are more marked in the case of the right vagus than in that of the left vagus and the sympathetic together. This shows that more depressor fibres are contained in the right vagus than in any one of the remaining nerves. Moreover, he observed that the increase in the outflow of blood is proportional to the depressor effect. In control experiments on rabbits, in which the depressor runs, as already stated above, not in the vagus trunk but as an independent nerve, he confirmed the view that these effects are really due to the action of the depressor nerve. Does such a relation exist between the stimulation of the vagus and the spleen volume? If such is proved, then we obtain a further proof in support of the view that this dilatation of the spleen is due to the depressor action, although it has already been confirmed by Bayliss himself in rabbits. In order to establish this point I took these four nerves as he did, and stimulated their central end separately. The result was exactly as was anticipated: the stimulation of the right vagus caused most conspicuous dilatation, less evident were the results of stimulating the left vagus or sympathetic, while the right sympathetic gave no result of this kind at all. Moreover, the dilatation provoked by the right vagus was nearly equal to the sum of those by the left vagus and sympathetic. In other words, the depressor fibres are pretty evenly distributed in both sides. The examples quoted below will show this relation. Exp. 1. 1. ii. 1926. The vagus and the sympathetic of both sides in the neck were prepared for stimulation. The central end of them was stimulated separately, the current being of the same strength. The results obtained are tabulated as follows: L. vagus + R. vagus L. sympath. L. vagus L. sympath. Increase of vol. (i) -6 c.c. *2 c.c. *5 c.c. *6 c.c. (ii) -6 c.c. -6 c.c. Decrease of vol. (after-effect) (i) (ii) 1-3 c.c. *8 c.c. *5 c.c. *7 c.c. 1-2 c.c. 1.1 c.c. Exp. 2. 8. ii. 1926. The same kind of experiment was done. The results are as follows L. vagus + R. vagus L. sympath. L. vagus L. sympath. Increase of vol. (i) 1-5 c.c. *7 c.c. *8 c.c. (ii) 1-3 c.c. 1-1 c.c. -9 c.c. 1-3 c.c.
ACTION OF VAGUS ON SPLEEN. 299 These results undoubtedly show that what has been found by Bayliss in the submaxillary gland, is exactly applicable to the spleen, and in my opinion this is sufficient evidence that the stimulation of the central end of the vagus causes in cats the dilatation of the spleen, which action is duly attributable to the effect of the depressor nerve. However, in some cases, if the strength of the stimulation and the sensibility of the animal are brought into such a relation as to cause a strong sensory impulse without the depressor effect, then such a contraction as Roy observed might be seen. In this case there is of course no fall but rather a rise in blood-pressure. Therefore Roy's view that the vagus acts as a sensory nerve is still to a certain extent correct, especially since, in the tracing given by Bayliss(8) taken from rabbits in which the depressor nerve but not the vagus was stimulated, only the dilatation is seen and no after-contraction at all. SUMMARY. 1. The stimulation of the peripheral end of the vagus in the neck causes a diminution in the spleen volume. But if the fall of the bloodpressure is prevented by means of a blood-pressure compensator, or if the vagus is stimulated inside the thorax below the point at which the nerve sends out its cardiac branch so that no considerable change in blood-pressure occurs, the stimulation does not provoke any change in the spleen volume at all. Moreover, the spleen volume varies with the blood-pressure. Therefore this shrinkage of the spleen is merely a passive effect due to the fall of the blood-pressure. 2. The stimulation of the central end of the vagus provokes the dilatation of the spleen. This dilatation is to be ascribed to the stimulation of the depressor fibres, which in cats are contained in the vagus trunk in the right side and both in the vagus and the sympathetic in the left side: the distribution of the fibres is nearly equal in both sides. Therefore the effect evoked by the right vagus is nearly as strong as the sum of those by the vagus and the sympathetic respectively of the left side. I desire to express my gratitude to the late Prof. Langley, who kindly gave me the privilege of working in his laboratory, and my sincere regret for his death. My thanks are also due to Prof. Barcroft for his suggestions, encouragement and help continuously given to me throughout the whole course of this research.
300 T. MASUDA. REFERENCES. 1. Bulgak. Virohow's Arch. 69. p. 181. 1877. 2. Roy. This Journ. 3. p. 203. 1881. 3. Skramlik and Duran-Cao. Zeitsch. f. d. ges. exp. Med. 45. p. 460. 1925. 4. Boer and Caroll. This Journ. 39. p. 314. 1924. 5. Oehl. Schmidt's Jahresber. 141. p. 275. 1869. Cited from Bulgak, l.e. 6. Schafer and Moore. This Journ. 20. p. 1. 1896. 7. Roberts. Ibid. 55. p. 351. 1921. 8. Bayliss. Ibid. 37. p. 264. 1908.