(Received March 26, 1936) Mann[1925], Izquierdo and Cannon [1928], Barcroft and Stephens

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1 189 6I2.4I:621.8 i SOME OBSERVATIONS ON THE DENERVATED SPLEEN BY J. BARCROFT AND R. H. E. ELLIOTT1 (From the Physiological Laboratory, Cambridge) (Received March 26, 1936) WITHIN the last decade, investigations on the reactions of the denervated spleen to exercise, emotion, operative procedures, etc., have been variously carried out by de Boer and Carroll [1924], Hargis and Mann[1925], Izquierdo and Cannon [1928], Barcroft and Stephens [1927], and Barcroft [1930, 1932]. It was with the idea of confirming and amplifying the observations of these authors that the work described in this paper was undertaken. The general scheme of denervation has usually been the destruction of the nerves which pass into the spleen at the hilus. This method-has not always proved satisfactory, as shown by subsequent histological examination. Dr J. B. Gaylor has kindly made a histological examination by the Bielchowsky method of the denervated spleens of animals used in B a rcroft's experiments and reports that, in the spleen of " Tilley II ", there is no doubt that healthy nerve tissue is to be seen running through the muscular tissue of at least one of the trabecule of the organ, and, so far as he can form an opinion, the fibres themselves are ones which have been there during the whole time of the experiment. In order to ensure as complete a denervation of the organ as possible, it was decided to cut through the splenic artery and vein, and reunite the transected vessels with the aid of couplers described by Lim in It was hoped that in so doing we should at the same time sever any fine nerve fibres which we might have overlooked during the process of cleaning the vessels. METHOD The animals employed were bitches, and the operative procedure was done under C.E. mixture in two stages. At the first operation the abdomen was opened through a mid-line incision, the splenic vessels Member of the Department of Su'gery, Presbyterian Hospital, New York City.

2 190 J. BARCROFT AND R. H. B. ELLIOTT' exposed and cleaned throughout the greater part of their length, and all visible nerves supplying the spleen resected for a distance of about 2 cm. Using Lim's' aluminium coupler technique, the splenic artery and vein were transected and rejoined central to their division into anterior and posterior branches, thus effectively destroying any nerve filaments running in the adventitial coats of the vessels. The spleen was then restored to the abdominal cavity, and the wound sewn up. The second operation, which consisted of a simple exteriorization of the spleen according to the method described by Barcroft and Stephens [1927], was performed 24-3 months after the first. Complete degeneration of the nerve fibres, it seems, may have been realized, for in one animal which came to post-mortem shortly after its second operation, no vestiges of nerve filaments could be seen in histological sections of the spleen stained by the Bielchowsky silver method. As a control, a dog "Curley" was used which had had its spleen with intact nerve supply exteriorized at the same time as the other animals were undergoing their second operation. The post-operative treatment was the same as that advised by Barcroft and Stephens, as was also the celluloid and grease pencil technique of recording the surface area of the organ. The tracings thus obtained were transferred to paper and measured by means of a planimeter. SIZE OF THE SPLEEN Tracings of the spleen were begun in two instances at the time of operation, and in all cases by the second post-operative day. It will be seen from Fig. 1 that during the first few days after operation the organ undergoes rapid enlargement, and then, during the course of the next few days, an almost equally rapid shrinkage. This is especially pronounced in the denervated specimens ("Blackey" and "Tilley III ") the alterations in size during the first week being almost twice as rapid and extensive as in the case of the innervated one. The enlargement, as has been generally agreed, may in all cases be due to loss of tone and blood-pressure changes within the organ (particularly in the denervated preparations), but the rapid shrinkage that takes place during the next two weeks as well as the more gradual diminution in size over the next few months, still remain inadequately accounted for-especially where the denervated spleen is concerned. It is interesting in this connexion to note that at the time of the second operation all denervated spleens appeared somewhat enlarged in 1 We have to thank Prof. Lim, who kindly presented us with the couplers used.

3 SOME OBSERVATIONS ON DENERVATED SPLEEN 191 size, though of a normal colour. No scars or adhesions were encountered, and the surface of the organ presented a smooth, glassy appearance, probably due to loss of tone. Barcroft and Stephens, while investigating the extreme limits of the dimensions of the spleen, recorded the case of an 18-kg. dog in which I~ I _ 95 - Control 90 j -- "oblackey" 85 is-- is--a Tilley Im _ 70 b c45 O Days after exteriorization Fig. 1. Variations in size of the spleen in resting animals. this organ, following denervation and ligation of its venous return, attained a weight of 365 g. They compared this spleen with that of a 16-kg. dog killed under anesthetic by bleeding, whose spleen weighed 33 g. In pointing out the elevenfold difference between the two, they gave as their opinion that the great size of the spleen of the first-mentioned dog might not represent the extreme capability of the organ. Through the death of an animal on the day following its first operation we were able to confirm and amplify their observations. The dog in

4 192 J. BARCROFT AND R. H. E. ELLIOTT question weighed 16 kg. At post-mortem it was found that the coupler on the splenic vein had twisted, and the vessel was completely thrombosed. The spleen weighed 560 g., 50 g. more than the liver of the same animal, and probably 18 times as much as it would have weighed had its nerve supply been left intact and the animal died of haemorrhage. The above-mentioned authors, assuming the blood volume of their dog to be about 10 p.c. of body weight, and therefore from 1-6 to 1-8 kg., reasoned that at least 340 c.c., or about one-fifth of the entire blood of the animal was in the spleen. Continuing the analogy, and assuming the blood volume in our dog to have been 1-4-1*6 kg., and the amount in the spleen to have been about 535 c.c., the organ must have held at least one-third of the original blood volume of the animal. We do not know the extent to which this blood volume had been increased by dilution of the circulating fluid, but apart from such dilution the loss of one-third of the blood volume would probably have been a sufficient cause of death. EMOTION Hargis and Mann [1925] and Barcroft [1930] have pointed out that the extent of the splenic response to emotional stimuli may be largely governed by the disposition of the experimental animal, as is known to be the case with many other responses [Pavlov, 1927]. Barcroft emphasizes the difficulties of obtaining suitable animals for this type of experiment, and states that the extreme differences in temperament shown by the different experimental subjects are largely responsible for this difficulty. It so happened that two of our animals were possessed of very much the same type of disposition in that they were both extremely highly strung. Of these, " Curley " happened to be a control dog, and the other, "Blackey", a denervated preparation. "Tilley III", the second denervated spleen dog used in these experiments, was of a temperament diametrically opposite to the other two. The only stimulus employed was a sudden loud noise, made by unexpectedly striking a sharp blow on the top of the table on which the dogs were lying. To this the spleen of the control dog responded with an almost instantaneous, brisk contraction which was nearly as extensive as that caused by severe exercise. On the other hand, the contraction of "Blackey's" spleen was much less extensive, though she appeared outwardly more startled than the control. With "Tilley III" a definite change in the size of the spleen was obtained on only one occasion, when

5 SOME OBSERVATIONS ON DENERVATED SPLEEN 193 TABLE I. Change in area of spleen following emotional stimulus. Before After stimulus stimulus Change sq. cm. sq. cm. Diff. p.c. "Curley" 24 19* Av. 20 "Blackey" 41* Av "Tilley III" there was a 6 p.c. decrease'in surface area. She proved very hard to startle, no matter how loud or unexpected the stimulus. The results of previous workers on the denervated spleen have been discordant. Hou and Lim [1929] noted a contraction of the denervated spleen on excitement and were of the opinion that the secretion of adrenaline was chiefly responsible for the contraction of this type of preparation. Hargis and Mann, on the other hand, found that the denervated organ did not contract when their animals were suddenly frightened. Our results indicate that marked differences in response occur in different individuals, the excitable "Blackey" giving a well-marked contraction while "Tilley III" gave little or none. EXERCISE The exercise consisted of running variable distances. For light exercise the animals were usually run once up and down a corridor and covered a distance of m. in so doing. For more strenuous exercise the dogs were run up two flights of stairs, around a large room from four to six times, and then back down the two flights. The distance covered varied from 145 to 220 m., exclusive of the ascent and descent of the stairs. All distances were covered with the individual who was exercising the dogs running at top speed. The results are given in Table II. It will be seen that the results corroborate those of Hou and Lim and Barcroft and Stephens in that the denervated spleen underwent a contraction after this form of exercise. There was little difference&in the degree of contraction in " Blackey" and the control, but in "Tilley III" the contraction was less marked. PH. LXXXVII. 13

6 194 J. BARCROFT AND R. H. E. ELLIOTT TABLE II. Change in area of spleen following exercise Area Area before after Distance metres exercise sq. cm. exercise sq. cm. Diff. Change p.c. Average change "Curley" stairs ) stairs stairs "Blackey" ) "Tilley III" EFFECT OF TEMPERATURE Barcroft [1925] states that the increase in blood volume which occurs at high temperatures led him to start his investigations of the role of the spleen as a blood depot. We therefore decided to ascertain what effects exposure of our dogs to extremes of temperature would have on the size of their spleens. Heat. As it was deemed necessary to determine first the effect of heat applied directly to the spleen, wads of cotton wool soaked in water warmed to from 59 to 650 C. were applied directly to this organ and maintained in position for about 1 min. Tracings were taken immediately before and after this procedure, and though the experiment was repeated on at least three separate occasions with each animal, we could detect no alteration whatever in the size of the spleen. Following this, the animals were put separately into a chamber which had been previously warmed to temperatures ranging from 47 to C., about the same degree of heat as is often met with in the tropics. As the chamber possessed plate-glass walls, a close watch could be kept

7 SOME OBSERVATIONS ON DENERVATED SPLEEN 195 both on the temperature inside and the dogs. The temperature showed very slight variations, tending to rise only a degree or so during the course of each experiment. The dogs all reacted normally to the heat, and after a few minutes' exposure, ceased walking about and lay down, panting. When the allotted time was up, the chamber was entered with the least possible disturbance, and tracings of the spleen obtained. The animals showed little tendency to move about when we entered, and were quite quiet while records of their spleens were being taken. TABLE III. Change in area of spleen following exposure of animal to heat Temp. Length Change Area Area of of in before after chamber exposure body exposure exposure Change OC. min. temp. 'C. sq. cm. sq. cm. Diff. p.c. Control * "Blackey" * "Tilley III" * * As will be seen from Table III, the spleen of the control animal underwent a distinct shrinkage in every experiment. On the other hand, the denervated spleens appeared distinctly larger, redder, and more turgid than under normal resting conditions, and increments in size up to 11 p.c. were noted. These results would seem to indicate that the stimulus to contraction of the spleen in the normal dog under these circumstances is principally a nervous one. Cold. Stephens, in some unpublished investigations performed in this laboratory in 1927, found that the innervated dog's spleen did not respond to the direct application of cold, but underwent a considerable diminution in size when the animal was exposed to cold air. We were able to corroborate his findings, and extend them to the denervated spleen also. Ice packs were applied directly to the spleens of our dogs, and held in place for a minute or longer. Tracings taken immediately before and after the experiment revealed no alterations in size in either the normal or denervated preparations. Several repetitions of this procedure on different days gave like results.

8 196 J. BARCROFT AND R. H. E. ELLIOTT Through the courtesy of the late Sir William Hardy, Director of the Low Temperature Research Station at Cambridge, we were allowed to use one of the 00 C. rooms for our investigations. The procedure was a simple one, and consisted merely in registering the resting surface area of the spleen at a room temperature of about 180 C., then putting the animal in the 0 C. chamber for 20 min., and at the end of this time taking a tracing while the dog was still in the chamber. Someone always remained with the animal while she was in the cold room, and though all the dogs showed a natural tendency to move about in order to keep warm, they were not unduly excited or active. The results were somewhat marred by the fact that on the day these experiments were started the control dog came on heat and the size of her resting spleen was therefore somewhat diminished [see Barcroft and Stephens, 1928]. Nevertheless, the organ definitely contracted (see Table IV, first and second TABLE IV. Changes in area of spleen following exposure of animal to cold Temp. Length Change Area Area of of in body before after chamber exposure temp. exposure exposure Change OC. mm. OC. sq. cm. sq. cm. Diff. p.c. Control "Blackey" *5 23' "Tilley III" experiments) when the animal was exposed to cold. The experiment was repeated 5 months later (Table IV, third experiment), and a good contraction was given. The spleens of the denervated dogs also underwent decrements in size which, in the case of "Blackey", a short-haired animal, were more pronounced than in the control animal. The denervated spleen of " Tilley III ", however, who had long shaggy hair and was therefore better protected from the cold, showed less marked alterations in size, but it must be remembered that this animal gave also a much less marked response to emotional stimuli, exercise and heat. From these results it would seem that the contraction response of the spleen to cold is largely humoral in nature, and probably caused by the increased output of adrenaline which was shown to follow exposure of the organism to low temperatures by Cannon and his co-workers [1927].

9 SOME OBSERVATIONS ON DENERVATED SPLEEN 197 SUMMARY 1. In resting dogs the alterations in size of the denervated spleen during the first week after exteriorization are considerably more rapid and extensive than in the innervated organ. 2. A spleen deprived of its nerve supply and with its venous return cut off may hold as much as one-third of the original blood volume of the animal. 3. The denervated spleen may respond by contraction to the emotional stimulus of a sudden loud noise, but the contraction is considerably less than in the innervated organ. 4. Exposure of dogs to high temperatures results in a contraction of the innervated spleen, and in a slight enlargement of the denervated organ. 5. Exposure of dogs to low temperatures brings about a contraction in both the innervated and denervated spleen. REFERENCES Barcroft, J. (1925). Lancet, 1, 319. Barcroft, J. (1930). J. Physiol. 68, 375. Barcroft, J. (1932). Ibid. 76, 436. Barcroft, J. and Stephens, J. G. (1927). Ibid. 64, 1. Barcroft, J. and Stephens, J. G. (1928). Ibid. 66, 32. Cannon, W. B., Querido, A., Britton, S. W. and Bright, E. M. (1927). Amer. J. Physiol. 79, 466. de Boer, S. and Carroll, D. C. (1924). J. Physiol. 59, 312. Hargis, E. H. and Mann, F. C. (1925). Ibid. 75, 180. Hou, H. C. and Lim, R. K. S. (1929). Lignian Sci. J. 8, 301. Izquierdo, J. J. and Cannon, W. B. (1928). Amer. J. Physiol. 84, 545. Lim, R. K. S. (1927). Chinese J. Physiol. 1, 37. Pavlov, I. P. (1927). Conditioned ReJfexes. Trans. and edit. Anrep. Oxford.