corresponding to the pulsatile flow of blood normally produced by

Transcription

1 THE HEART, LUNG, KIDNEY PREPARATION. BY F. A. BAINBRIDGE AND C. L. EVANS. (From the Institute of Physiology, University College, London.) THE methods hitherto generally employed for the perfusion of isolated mammalian organs are attended by certain drawbacks among which may be mentioned first the difficulty of ensuring adequate oxygenation of the blood and removal of carbonic acid, and secondly the absence of anything corresponding to the pulsatile flow of blood normally produced by the heart. In the case of some organs these drawbacks are not of great importance. The kidneys however are so sensitive to lack of oxygen, and probably also to the absence of the intermittent driving force of the heart, that perfusion of the isolated kidney has not yielded results which could justly be compared with those obtained in the living animal. The heart-lung preparation devised by Knowlton and Starling(5) seemed to offer a means whereby these drawbacks might be obviated, and the kidneys might be perfiused either in situ, or after removal from the body under conditions closely approximating to the normal; and the present paper deals mainly with the method employed to carry out such perfusions. The method may be described as a heart-lung-kidney preparation. Methods. Two dogs are used for each experiment-one for the heart-lung preparation and one for the kidney or kidneys. After a preliminary dose of morphia the dogs are ansesthetised with chloroform and ether. The heart-lung preparation is made from one dog according to the method of Knowlton and Starling (5). Simultaneously the kidney preparation is made on the other dog by one or other of the two following methods, and finally the heart-lung and the kidney preparations are connected. (1) By this method the kidneys are left in situ and either one or both may be used for the perfusion experiment. After the removal of

2 HEART, LUNG, KIDNEY PREPARATION. 279 the greater part of the small and large intestines the aorta and vena cava are tied off a short distance below the origin of the renal vessels. The suprarenal veins are next tied off and the suprarenal glands are put out of circulation so far as possible by tying ligatures round them. Ligatures are then placed round the aorta and the vena cava just above the origin of the renal vessels but they are not tied. The next and most important step consists in tying off all the vessels arising from the aorta and vena cava between the upper and lower ligatures just mentioned except the renal arteries and veins. This is best effected by raising the left kidney from its bed so as to expose the posterior aspect Fig. 1. of the aorta and vena cava from which arise many small lumbar vessels, one at least being quite close to the origin of the renal arteries. Any considerable leakage will interfere with the success of the subsequent experiment, and it is essential to occlude these vessels as completely as possible. If this part of the operation involves much handling of the left kidney it is desirable to ligature it off and to use only the right kidney for the experiment.

3 280 F. A. BAINBRIDGE AND C. L. EVANS. The aorta and vena cava are then clamped just below the renal vessels and cannulae are placed in them below the clamps and are connected with the heart-lung preparation as shown in Fig. 1. The cannulae contain a little hirudin solution and a thermometer is placed in the arterial cannula A. Up to this point of course the kidneys are receiving their blood supply by the normal route. Finally the cannula A is filled with blood from the heart-lung preparation, the clips on the aorta and vena cava are removed, and the blood is allowed to pass upwards along the aorta through the kidneys. At the same moment the ligatures B and C, previously passed round the aorta and vena cava, above the renal vessels are tied off, and the kidneys henceforth receive their blood supply solely from the heart-lung preparation. With care the transition from the normal blood supply to that from the heart-lung preparation can be effected without any pause in the flow of blood through the kidneys. As soon as the preparation is completed the spinal cord is divided in the neck, and is destroyed by passing a rod down the vertebral canal; this measure is necessary in order to cut off the kidneys from the central nervous system and to prevent asphyxia of the kidneys by vaso-constriction when the dog is killed. The kidneys (or kidney) may now be freed fromn their fatty covering and any bleeding points touched with the cautery; loss of blood from this source is usually trivial. Cannule are then placed in the bladder or ureters. The reversal in the direction of the blood flow along the aorta and vena cava does not seem to interfere in any way with the rate of flow through the kidneys and the circulation appears perfectly normal. Prov'ided the kidneys are not exposed and are kept warm the preparation will last for some hours-in fact as long as-the heart and lungs hold out. (2) By this method one kidney is removed from the body and perfused as an isolated organ. After the removal of the greater part of the small and large intestines a ligature is passed round one renal vein close to its junction with the vena cava and is left loose. The kidney on that side is gently raised from its bed, the renal artery is exposed for j-1 of an inch and a ligature is passed round it. As soon as the heart-lung preparation is ready the cannula A (Fig. 2) which is to be inserted into the renal artery is filled with hirudinized blood and a screw clainp B is so adjusted that a slow but steady stream of blood is flowing from the cannula.

4 HEART, LUNG, KIDNEY PREPARATION. The renal artery is then occluded by a clip near its origin, the artery is opened beyond the clip and the cannula is at once inserted and tied in the artery. At the same moment the other operator ties off the renal vein close to the vena cava. Immediately the ligatures are tied, the renal vein is divided to allow the blood to escape and the screw clamp B on the tube conveying arterial blood from the heart-lung preparation is loosened. The kidney is rapidly removed from the body, placed in a funnel and supported on gauze. The blood escaping from the renal vein 281 Fig. 2. passes down the funnel and into the reservoir C. The funnel contaitning the kidney is covered by a piece of oile(d silk to prevent evaporation. A cannula may be placed at leisure in the ureter. The general arrangement of the complete preparation is shown in Fig. 2. It has been found that the transition from the normal circulation to that provided by the heart-lung preparation occupies about a minute PH. XrLVIII. 19

5 2i82 P. A. BAINBRIDGE AND C. L. EVANS. including the tying of the ligatures, but the period during which the kidney is deprived of arterial blood is less than i minute since the arterial blood enters the kidney directly the cannula is placed in it, and before it is actually tied in. In order to avoid delay in inserting the cannula into the renal artery and establishing the new circulation it is desirable to ascertain beforehand that the arterial cannula is of suitable size. The rate of flow of blood through the kidney is determined by opening the clip D on the side tube of the funnel and pinching the rubber connection below it. The blood supply to the kidney could if necessary be warmed by passage through a small glass spiral immersed in a bath at suitable temperature, but in our experiments the circulation was so rapid that we did not find this arrangement essential. This method may at first sight appear less satisfactory than the first since it involves more handling of the kidney. So far as our limited experience goes, however, gentle handling of the kidney, provided it is kept warm, does not seem to interfere with its functional capacity. This method has the advantage also that the whole of the blood leaving the kidney can be collected, whereas by the first method some leakage from small branches arising from the aorta or vena cava is unavoidable and may be sufficient to interfere with the success of the experiment. Applications of the Method. As yet we have made three successful experiments only, and our primary object has been to test the efficiency and possibilities of the preparation. The points which have been examined are (1) the gaseous metabolism, (2) the blood flow, and (3) the flow of urine; and the general results are shown in the two following protocols (p. 283). Analysis of urine. Three c.c. of urine in all were collected. It was acid in reaction, yellow and turbid, and contained phosphates, chlorides, sulphates and a trace of protein. It contained also 1 0/0 of nitrogen in the form of urea and ammonia: this was estimated by Dr Plimmer using the urease method. Microscopic examination showed some cells from the pelvis of the kidney, very few red cells and many fat droplets (the two latter being possibly accidentally introduced in the insertion of the cannula into the ureter). Gaseous metabolism. The gaseous metabolism was determined in successive periods of 20 minutes by the method employed by Evans (4) for the study of the metabolism of the heart-lung preparation. In one experiment the metabolism of the heart-lung preparation alone was first determined, and then that of the same preparation with the

6 HEART, LUNG, KIDNEY PREPARATION. 283 I. The preparation was made by the first method, the kidneys being kept in 8itU. One kidney was used. Heart-lung preparation ready at 2.30, and the preparation completed at Weight of heart=37 grms.: weight of kidneys=30-5 grms. Temperature of blood entering heart was 3650 C.: pulse rate 154 per minute. Output of heart=395 c.c. per minute. Blood-pressure remained constant at 100 mm. Hg. Temperature of blood entering kidneys= C. No uirine secreted. Gaseous metabolism of heart-lung preparation was first determined, and subsequently that of heart-lung-kidney preparation: the figures for the metabolism of the kidney are determined by difference. The observations were made by Evans' method(4). 02 used 02 used CO given Blood-flow Total per per 1 grm. oif per Total through c.c. c.c CO hour byy of kidney hour by Respi- kidney 02 used given ok kidney hourly kidney ratory in Preparation Time c.c. per per hour per hour c.c. c.c. c.c. quotient 1 min. Heart-lung , Heart-lung Not kidney observed The rate of the blood-flow through the kidneys was varied by means of a screw clamp on the arterial tube to the kidney. Mean respiratory quotient of kidney=0-71. Average 02 eonsumption by kidney=0-046 c.c. per 1 grm. per minute. II. The preparation was made by the second method, and one kidney was used. Weight of heart=79 grms.: weight of kidney=35-5 grm. Only oxygen consumption was measured, and cndema of the lungs prevented any direct observation at the end of the experiment of the oxygen used by the heart. The oxygen consumption of the heart and lungs was calculated from previous figures (obtained by E vans) at 3-08 c.c. 02 per 1 grm. per hour. Preparation Heart-lungkidney Time Remarks Urine movement in cms. along scale Normal 0,, add to blood 2 c.c. 500/0 NaSO4,, c.c. NaSO Total 02 used per hour C.C. 393 Calc. 02 used by 02 used by kidney kidney per per 1 grm. per hour hour c.c. c.c Rate of blood-flow in kidney in cc. per 1 min The urine flowed along a tube provided with a scale graduated in centimetres. It flowed along the tube in spurts synchronous with visible contractions of the pelvis of the kidney and the ureter; these contractions took place at the rate of about 12 per minute. 19-2

7 284 F. A. BAINBRIDGE AND C. L. EVANS. addition of the kidneys, the metabolism of the kidney being therefore estimated by the difference. The oxygen consumption of the kidneys in these two experimnents during the period when no urine was being secreted was fairly constant; the average consumption per grm. of kidney per minute being 0046 c.c. in the first, and c.c. in the second experiment. These figures are quite comparable with those obtained by Barcroft and Brodie(1) on the dog, which varied from 0,008 to c.c. and with those of Barcroft and Straub(3) which varied froin 0 03 c.c. to 0 1 c.c.; in both these sets of experiments some urine was being formed by the kidneys. In the first experiment the effect of varying the rate of blood flow through the kidney upon the consumption of oxygen was investigated. It will be seen that with a flow varying from 12J-55 c.c. per minute the oxygen consumption over a period of 20 minutes showed very little variation. This result confirms the observation of Barcroft and MUller(2) on the submaxillary gland that the oxygen consumption of a tissue is determined primarily by the needs of the tissuie and not by the blood supply, provided that the latter reaches or exceeds a certaini necessary minimum. In the second experiment in which the addition of sodium sulphate evoked an immediate flow of urine the consumption of oxygen reached 0-07 c.c. per grm. per minute, a figure quite comparable with those obtained by Barcroft and Brodie, and by Barcroft and Straub with the same diuretic. In one of Barcroft and Brodie'so) experiments the injection of sodium sulphate increased the oxygen consumption from to c.c. per 1 grm. per 1 minute. Unfortunately, in another experiment in which urine was obtained when the blood was diluted with Ringer's solution the oxygen consumption of the kidneys was not determined. The output of carbonic acid and the respiratory quotient varied considerably in successive periods of 20 minutes, these variations being no doubt due to different rates of washing out of 002 by the circulating blood, but the mean quotient in Exp. 1 is not very different from that of the heart, and is probably fairly accurate. Since the figures for the oxygen consumption of the kidneys are obtained indirectly, their accuracy will be affected by definite alterations in the work and consequently the gaseous metabolism of the heart, and it is necessary to guard against this source of error. Evans has shown, however(4,6), that the average oxygen consumption of the beart-lung preparation apart from the presence of adrenalin, or violent changes of blood-pressure, is fairly constant; and

8 HEART, LUUNG, KIDNEY PREPARATION. 285 the accuracy of our method could easily be checked by direct observation of the oxygen consumption made simultaneously with Barcroft's apparatus. In the two experiments just described these sources of error could be practically disregarded, and the results show that the gaseous metabolism of the kidneys in the heart-lung-kidney preparation is similar in amount to that observed by other methods for the kidneys in the body. The blood flow. The amount of blood flowing through the kidneys can be varied at will, and depends upon the relative resistance of the renal vessels, and of the artificial resistance introduced into the heartlung preparation. In our experiments, this resistance amounted to about mm. of Hg., and the general arterial pressure was about 100 mn. Hg. By tightening a screw clamp on the tube supplying blood to the kidneys the rate of flow through the kidneys could be diminished at will. In the second experiment in which the pressures were those just mientioned the rate of blood flow through the kidneys was 80-98c.c. per min. In Barcroft and Straub's(3) experiments the flow varied from c.c. per min. in the absence of any diuretic. In our experiments owing to the loss of vasomotor control the renal vessels would possess little or no tone. The flow of urine. The flow of urine may be regarded as the fundamental test of the success of the preparation. In two of our experiments urine was readily obtained, in one case after dilution of the blood with Ringer's solution, and in tlle other after the addition of sodium sulphate to the blood. In none of the three experiments' was a spontaneous flow of urine obtained as soon as the preparation was started. We were at first inclined to attribute the absence of urine at the outset of the experiment either to the effect of the anaesthetic on the kidney, or to the experimental procedure itself. It must be remembered, however, that in these experiments the blood is rather concentrated, and that it remains practically constant in composition since it does not receive urea or other tissue products. It seems possible therefore that the kidney does not form urine simply because the blood is not providing an adequate chemical stimulus. The readiness with which urine is formed when the blood is diluted, or on the addition of a diuretic, favours this view, but further investigations will be made. The action of sodium sulphate (protocol II) was interesting in that it evoked a prolonged flow of urine without appreciably altering either the concentration of the blood or the rate of flow through the kidney.

9 286 F. A. BAINrBRIDGE AND C. L. EVANS. It did, however, notably increase the gaseous exchange, and the analysis of the urine showed that the latter was a true secretion. On the whole our observations entirely confirm and agree with those obtained by other workers using different methods, and show that the heart-lung-kidney preparation will yield results comparable with those obtained in the living animal. It seems fair to conclude then that the preparation is a reliable one and that the kidneys are in a normal functional condition. The advantages of the preparation are, first, that the blood-pressure and the rate of blood flow through the kidney are under control, and can be varied at will, and, secondly, that the kidneys are practically freed from the ever varying influence of the tissues, and that the composition of the blood can be kept constant, or can be altered as desired. The preparation offers, therefore, a means of investigating independently the part taken on the one hand by circulatory changes, and on the other hand by the composition of the blood bringing about the flow of urine. SUMMARY. A method for perfusing the isolated kidney with arterial blood from the heart is described. Under these conditions the arterial blood-pressure, blood flow and temperature are under control, and the gaseous exchanges of the kidney can be investigated. The kidney thus isolated is capable of secreting urine. The gaseous metabolism of the isolated kidney is increased when the urine secretion is provoked by the aid of diuretics such as sodium sulphate. The respiratory quotient of the kidney is similar to that of the heart. The gaseous metabolism of the kidney is not altered by changes in the rate of blood flow through the organ. The expenses of this research have been defrayed by a grant from the Royal Society. REFERENCES. (1) Bareroft and Brodie. This Journal, xxxiii. p (2) BarcroftandMuller. Ibid. xuv. p (3) Barcroft and Straub. Ibid. XLI. p (4) Evans. Ibid. XLV. p (5) Knowlton and Starling. Ibid. xuv. p (6) Evans. Ibid. XLviI. p