comparable with the normal resting condition of the organ in the unopened contraction, to determine the haomoglobin percentage of the blood, and

Transcription

1 ON THE OUTPUT OF HAEMOGLOBIN AND BLOOD BY THE SPLEEN. BY E. W. H. CRUICKSHANK. (From the Physiological Laboratory, Cambridge.) BARCROFT'S(1) experiments on the protective effect of the spleen suggest that the spleen, contracting in response to stimuli reaching it from the central nervous system by way of the splanchnic nerves, furnishes the body with an increase in red blood cells. It seemed, therefore, of value to determine the haomoglobin content of the blood expelled from the spleen during contraction and to secure by direct methods further data upon the blood content of the spleen. Methods. The aim was to measure the contraction of the spleen, to collect the blood from the spleen and to estimate its rate of flow during contraction, to determine the haomoglobin percentage of the blood, and to relate this to the curve of contraction. In a few preliminary experiments a plethysmograph was used in order to secure records of the change in volume of the spleen, but it was soon found that the manipulation necessary to place the spleen in a plethysmograph, however gently it may be handled, rendered the results valueless; any exposure of the spleen, far more any handling of it, placed it immediately in a condition which could not be regarded as strictly comparable with the normal resting condition of the organ in the unopened abdomen. Anyone who has a surgical experience of the spleen knows how sensitive it is to environmental changes. To free it from its omental anchorage by tying any of its numerous vessels, to expose it, even, is to stimulate it. The plethysmograph was discarded, and in the experiments here recorded the spleen was not exposed at all. The animal was kept warm by means of an electric table, the ansesthetic used was chloralose, 041 grm. per kilo for a dog, 0 07 grm. per kilo for a cat. All the experiments, the results of which are detailed in this paper, were performed on cats. Operation. The animal, dog or cat, is placed on its right side and a longitudinal incision about 10 cm. long and about 6 or 7 cm. from the mid-dorsal line is made, commencing over the costal margin. When the peritoneum has been cut the rib margin is retracted and a clip placed on the stomach at the posterior edge of the gastro-splenic omentum. Another

2 456 E. W. Hi CRUICKSHANK. clip is placed on the mesentery above the region of the superior mesenteric vein; the latter clip secures the mesentery to the abdominal wall, and thus when traction is made upon these two clips an excellent view of the area for dissection of the splenic artery and vein and the right splanohnic nerve is obtained. Should the dorsal end of the spleen tend to curl forwards into view, a third small clip is used to secure the mesentery to the abdominal wall, and thus the spleen can be kept absolutely under cover throughout the whole experiment; in fact it need never be seen at all. To obtain a curve of the contraction of the spleen without a plethysmograph the rate of expulsion of blood from the spleen during its contraction must be determined when the circulation is stopped, and here one must remember that the spleen is partly supplied by blood through the right gastro-epiploic artery. By means of blunt dissection the coeliac axis and the junction of the splenic, mesenteric (usually two), and portal veins are cleared (see Fig. 1). A cannula is placed in the superior mesenteric vein. L. Gasrro-Epiploic a..>. -- -~~~~~K ~~~~R. Gastro-Epipl~oic X R Sz ~~~~takc a. a. Splenic arrery Gastric a. t -SUP. Afesenl'eric veln.sup.mesenrer,c vein: --! \\/7 Coeliac axis Fig. 1. Sup. Mesenteric artery= Portal vein. R Gastric a Aot Diagrammatic representation of the splenic and other vessels exposed in the dissection described in the text. A 5 or 10 c.c. microburette, which has been washed out with saturated potassium oxalate and dried in a current of warm air, is attached at its upper end to the venous cannula, a shielded electrode is placed on the splenic artery, clips are placed in quick succession on the portal vein and coeliac axis' and blood allowed free access to the burette. The nerves l This ensures that no collateral circulation will take place by way of the gastro. epiploic branch of the hepatic artery.

3 Hi.EMOGLOBIN IN SPLEEN. on the splenic artery are now stimulated with a weak induction current and the rate of expulsion of blood timed on a moving drum with a Jaquet clock marking fifths of a second. I. THE RAEMOGLOBIN CONTENT OF SPLENIC BLOOD. Three methods were open for the estimation of haemoglobin, the hsemoglobinometer, the colorimeter and the determination of the oxygen capacity of the blood. To find which method would give the most consistent results, several comparative estimations were made on cats' blood of varying haemoglobin strength. Taking the reading for normal arterial blood determined by the Duboscq colorimeter as 100, the average of the results obtained is as follows: Haldane Duboseq Barcroft hemoglobinometer colorimeter manometer Arterial blood, carotid ( Venous blood, spleen resting Venous blood, spleen with splanchnic stimulation Difference In using the colorimetric method 1 c.c. of blood measured from the microburette was added to 1000 c.c. of distilled water. Since the colorimetric method agreed well with the other two and was least liable to experimental error, it was used throughout. The hemoglobin percentage varies markedly in different animals, and for the sake of facility in reading all haemoglobin percentages have been given in terms of the normal standard of 100. This first series of experiments, both on cats and dogs, was carried out primarily to determine to what extent the spleen may act as a reservoir for hemoglobin, not only a reservoir for blood, and incidentally thereby able to add to the hemoglobin content of the body, but a reservoir in the sense that the blood stored in the spleen has a heemoglobin content in excess of the normal. It was argued that if the spleen be a reservoir for heemoglobin, then it should be possible to show an increase in the heemoglobin content of the blood expelled from the spleen during contraction. In the first four experiments upon cats the splenic artery was not clamped; the normal rate of flow was first determined, and then the rate of flow during and after stimulation of the branches of the left splanchnic nerve lying upon the splenic artery. The blood was collected in the microburette, the rate of flow for each successive c.c. being recorded by a signal; it was then run into distilled water contained in a series of six one-litre graduated flasks, 1 c.c. being placed

4 458 E. W. H. CRUICKSHANK. in each flask, and the haemoglobin percentage determined colorimetrically. From Table I, which gives the details per c.c. of three experiments, it TABLE I. Showing relation of the percentage hiemoglobin content of the splenic blood (carotid blood=100 p.c.) upon contraction of the spleen, to changes in the output of blood. Exp. 1 Exp. 2 Exp. 3 Output Output Output in c.. in c.c. in c.c. Splenic blood: venous Hb p.c. per min. Hb p.c. per min. Hb p.c. per min. (a) Before stimulation st C.c nd,, 113* rd,, (b) During stimulation J 4th,, of splanchnic 5th,, nerves 6th,, th,, th,, will be seen that the rate of blood flow through the spleen increases rapidly upon stimulation of the splanchnic branches surrounding the splenic artery. This increase is maximal between the second and fourth c.c., after which it steadily declines, and when vaso-constriction is maximal the rate of flow becomes markedly less than the normal. According to B arcroft''s work the withdrawal of 10 c.c. of blood causes little or no alteration in the volume of blood in the general circulation, despite the fact that this amount constitutes a fairly large loss of blood for a cat of 2-3 kilos. In Exp. 3 it will be seen that at the eighth c.c. the blood velocity is reduced to one-third of the normal. If then the spleen is to expel its stored haemoglobin one would expect it to do so long before its blood supply had become restricted, in fact one would expect that the period of increased output, aided by contraction of the spleen, would show the greatest increase in haemoglobin. A glance at Table I and Fig. 2 shows such a surmise to be correct, and shows also that the greatest output of hwemoglobin takes place with the third and fourth c.c. In order to form a conception of the extent and rate of contraction of the spleen, one must determine the rate of expulsion during a period in which no blood is allowed to enter the organ from the circulation. To measure the rate of flow the portal vein and cceliac axis are clamped as nearly as possible at the same moment, the splanchnic nerve stimulated and the blood collected as previously described. Here, as in previous experiments, the relation of blood flow, or, as in this case, the rate of contraction of the spleen to the hwemoglobin output is clear; the hiemo-

5 H,IMOGLOBIN IN SPLEEN. 459 globin concentration increases with the contraction of the organ, being maximal approximately at the period of maximal contraction; this is _ A bo II I II1, cc Fig. 2. Lower curve, percentage hemoglobin in successive c.c. output from the spleen on splanchnic stimulation, for comparison with upper curve showing simultaneous increase in rate of blood flow. Average of results in Table I. shown in Table II and Fig. 3. Here again the greatest concentration of haemoglobin is associated with the third and fourth c.c. of blood and shows an increase which may amount to 40 p.c. (Exp. 1, Table II). Many comparative estimations have been made on the number of red blood cells in the splenic artery and vein. Several workers have found a diminution of red cells in the venous blood of the spleen, others an increase. Paton, Gulland and Fowler(2) came to the conclusion that there was no difference. On compression of the spleen they stated that the number of the red cells was decreased, but the method used for expelling blood from this organ was a very abnormal one. From the results which we have obtained one must conclude that the contraction

6 460 E. W. H. CRUICKSHANK. of the spleen does produce an increase in the number of red blood cells, that is, it increases the haemoglobin percentage of the blood. TABLE IT. Showing relation of the percentage heemoglobin content of the splenic blood (carotid blood = 100 p.c.) upon contraction of the spleen, to the rate of contraction, as shown by the velocity of the blood expelled, during cessation of the circulation in the spleen. Exp. 1 Exp. 2 Exp. 3 Output Outpuit Output in c.c. in c.c. in c.c. Splenic blood: venous Hb p.c. per min. Hb p.c. per min. Hb p.c. per min. (a) Before stimulation I1st c.c *0 (b) During stimulation 920 2nd, * *2 of splanchnic <,3rd, 138* * nerves 4th,, * t5th,, 110*0 1*5 * $4 10 a4 5 C) 0 n ; 4) ; 130 :4, a.0 to o 120 S 1101 cc Fig. 3. Lower curve, percentage haemoglobin in successive c.c. output from the spleen on splanchnic stimulation for comparison with upper curve showing simultaneous change in volume of the spleen, the circulation being stopped. Average of results in Table II. II. THE SPLEEN AS A RESERVOIR FOR BLOOD. D e Boer and Carroll(3), by the plethysmographic method, showed that in an animal breathing weak CO mixtures (0.92 p.c.) the spleen con-

7 H.dEMOGLOBIN IN SPLEEN. 461 tracted 2-3 c.c. in 26 minutes, while with stronger CO mixtures (3.0 p.c.) it contracted in volume to the same extent in approximately 3 minutes. Bar croft has clearly shown that the contraction of the spleen in the body is of an order much greater than that shown by the plethysmograph. The results of the experiments just quoted and those shown in Table III TABLE III. Showing time contraction of the spleen and the output of blood upon stimulation of the splanchnic nerves accompanying the splenic artery. Amount of blood expelled in c.c Exp. 1. Time (secs.) Exp. 2. Time (secs.) Av. Time (secs.) bear this out. Two experiments were carried out in which, under very weak faradic stimulation of the nerves accompanying the splenic artery, the spleen was allowed to contract until no more blood was expressed. A total of 9-5 c.c. and of 13-5 c.c. of blood was obtained in cats weighing 3 and 3-2 kilos respectively. The time for the expulsion of blood per c.c. is seen in Table III. The average of these results up to the ninth c.c. is plotted as a curve of contraction in Fig. 4. The curve in Fig. 3, which represents graphically the rate of output of the blood with the splenic eci ~I I I secs. Fig. 4. uirve of contraction of the spleen upon splanchnic stimulation from data in Exp. 1, Table III.

8 462 E. W. H. CRUICKSHANK. artery clamped, indicates the rate of contraction in c.c. per minute: Fig. 4, however, indicates much more clearly the nature of the change in volume. It is rather striking that in both these cases (Table III) the first 5 c.c. should be expelled in 16 minutes: in the first case, 9 c.c. were expelled in 4-8 minutes, while in the second, 9 c.c. required only 3-8 minutes. In this latter case 13-5 c.c. of blood were forced from the spleen in 7*8 minutes. In these cases, as in the others quoted, the period of greatest activity, namely, during the second to fifth c.c., is the period of greatest haemoglobin output. Discussion of results. Since the measurements of the rate of contraction are made on an organ which has been deprived of its oxygen supply for 5-7 minutes, these experiments give no information as to the normal rate of contraction with the circulation intact. Further, from such curves as are here given, no one would conclude that the contraction of the spleen is necessarily a uaiform movement in the reduction of its volume. These results simply indicate that upon splanchnic stimulation the spleen, in cats of 2-3 kilos, will deliver up an amount of blood varying from 7 to 13-5 c.c. In this last case the amount of blood produced by the spleen was exactly twice the post-mortem weight of the spleen in grams, a fact which is of interest in view of B arcroft''s statement(1) that "the post-mortem weight of the spleen may be taken as a minimum estimate of the volume of blood it drives into the circulation during strong exercises." The mechanism of this function has not, however, been demonstrated. It may be that the steady tonic contraction may be a basal movement having superposed upon it rhythmic movements of contraction and dilatation, the former dominating the latter. Roy(4) demonstrated normal rhythmic movements in the spleen and Schafer and Moore (5) showed that in asphyxia these normal rhythmic movements are markedly increased. As to the power of the spleen to act as a reservoir of haemoglobin, the present work would indicate that the ha,moglobin concentration, which undoubtedly takes place there, is not of any considerable significance. For example, if, as is possible under the present method of experimentation, 8 c.c. of splenic blood contained haemoglobin percentages of 105, 125, 140, 130, 120, 115, 110 and 105 respectively per c.c., the amount of haemoglobin represented by these 8 c.c. of splenic blood when added to the circulation would be equivalent to an addition of 9-5 c.c. of normal blood. As an isolated action this is meaningless: as a possible continuous rhythmical activity related to all the functional activities of the organism it may not be without some significance.

9 HiEMOGLOBIN IN SPLEEN. In discussing this action of the spleen in concentrating haemoglobin we are faced with the possibility that the organs rich in muscle tissue may concentrate haemoglobin to a certain extent as a result of their activity, the loss of fluid taking place in the capillaries of the resting organ, and being increased by the contraction of the musculature which would force more fluid into the tissue and lymph spaces. We may ask therefore to what extent the slow blood flow in the non-functioning capillaries and the subsequent contraction of a skeletal muscle would tend to concentrate the blood. If it were found that any muscular organ concentrated blood to an extent comparable with the spleen, then the haemolymphatic structure of the spleen could not be regarded as peculiarly a reservoir of haemoglobin. The tongue offered not only a suitable muscular organ, but one with its motor and sympathetic nerve supply differentiated; it was therefore possible to determine the effect of muscle contraction uncomplicated by vaso-constriction. Two experiments were carried out in which both lingual arteries and one vein were clamped, and the hypoglossal nerve stimulated with a weak induction current. The results shown in Table IV were negative, indicating that haemoglobin TABLE IV. Showing relation of the hemoglobin content of the blood (carotid blood = 100 p.c.) upon contraction of the tongue of the dog, to changes in the rate of output of blood upon stimulation of the hypoglossal nerve. Exp. 1 Exp. 2 Output Output Venous blood from in c.c. in c.c. the tongue Hb p.c. per min. Hb p.c. per min. (a) Before stimulation (1st c.c (b) During stimulation 2nd,, of the 12th nerve 3rd,, (4th,, concentration is not a consequence of muscle contraction. A slight rise in haemoglobin percentage may be expected where, under pressure, fluid is forced slowly from the blood into tissue and lymph spaces. 463 SUMMARY. 1. A method is described for estimating, in cats, the blood and htemoglobin output of the spleen without exposure of the organ. 2. The fluid expelled from the spleen pulp is often richer in hsemoglobin than the blood of the general circulation. The increase of concentration is maximal, in cats, with the third c.c. expelled, amounting to from 20 to 40 p.c. of the normal.

10 464 B. W. H. CRUICKSHANK. 3. Corroboration is given to the fact that the spleen is a reservoir for blood. 4. The amount of blood which can be added to the general circulation by contraction of the spleen upon stimulation of the splanchnic nerves is of the order of 2*6-5-6 p.c. of the total blood volume of the animal; the amount so added may be equal to twice the post-mortem weight of the spleen in grams. 5. The rate of contraction of the spleen upon splanchnic stimulation is shown to be maximal during the expulsion of the first 5 c.c., which represents approximately one-half of the total amount expelled, and takes about 1.5 minutes. It then decreases rapidly, maximal contraction being reached in 5-8 minutes, depending upon the amount of blood expelled. 6. It has been shown that the capacity of acting as a reservoir for blood and of concentrating haemoglobin is a peculiar property of the spleen not shared by other muscular organs, e.g. the tongue. I should like here to express my indebtedness to Prof. B a r croft for introducing me to the work on the spleen, and to Prof. A. V. Hill for allowing me facilities for carrying out certain of these experiments in the Department of Physiology, University College, London. REFERENCES. 1. Barcroft et al. This Journ. 60. p Paton, Gulland and Fowler. Ibid. 28. p De B oer and Carroll. Ibid. 59. p Roy, C. S. Ibid. 3. p Schafer and Moore. Ibid. 20. p