(From the Department of Physiology, The University, Aberdeen.)

6I2.22.085.IX THE EFFECT OF EVISCERATION ON THE RESPIRATORY METABOLISM OF THE DECEREBRATE PREPARATION. BY I. A. ANDERSON, R. A. CLEGHORN, J. J. R. MACLEOD AD J. M. PETERSON. (From the Department of Physiology, The University, Aberdeen.) A PRELIMINARY examination of the decerebrate eviscerate cat has been made with a view to investigating some of the problems of carbohydrate metabolism, and it is hoped that for this purpose it may prove more suitable than the spinal eviscerate preparation. Results obtained with the spinal preparation are open to the criticism that they may be dependent to an indeterminate extent upon the nature of the artificial pulmonary ventilation, and upon the abnormally low blood-pressure and presumably poor circulation. The accurate adjustment of artificial ventilation to the altering respiratory needs of the preparation would appear to offer at the moment an unsurmountable difficulty which does not arise with the decerebrate preparation. The decerebrate non-eviscerate cat was first examined, and later compared with the decerebrate eviscerate preparation Ṁethod. After fasting for at least 12 hours the cat was rapidly anmesthetized with ether, a Y-shaped tracheal cannula inserted and the common carotid arteries tied. Decerebration was performed bytrephining the skull and removing the brain above a line of section across the middle of the superior corpora quadrigemina. The time elapsing from the beginning of an8esthesia till decerebration was effected was usually about 15 minutes. No aseptic precautions were taken. A thermometer was inserted under the skin at the axilla and the preparation allowed to rest lying on its side. About 45 minutes after decerebration it was placed on its back and, while lying quietly in this position, cannula for the recording of blood-pressure and for withdrawal of blood were inserted into the left and right common carotid arteries respectively. If any fluid was to be infused the cinnula of a constant infusion pump was inserted in the right external jugular vein. Small Pearce valves made from the gut of the rabbit were fitted to the branches of the Y-shaped tracheal cannula. The 26-2

392 I. A. ANDERSON AND OTHERS. size of these branches was adjusted approximately to the natural respiratory dead space. Evisceration. In a number of experiments evisceration was performed after two analysis periods had been completed (4 hours after decerebration), and in later experiments evisceration was performed I hours after decerebration (half an hour before analysis was begun). Evisceration was rapidly effected by placing artery forceps on the superior and inferior mesenteric arteries, on the cceliac axis, on the portal vein, hepatic artery and bile duct and on the lower ends of the cesophagus and colon. These were replaced by ligatures, and the stomach, intestines, spleen and pancreas removed, leaving the liver with the hepatic artery, portal vein and bile duct tied but open to the inferior vena cava through the hepatic veins. The kidneys and adrenals were left with their circulation intact. In order to give support to the diaphragm, the abdomen, after evisceration, was lightly packed with cotton-wool wetted with warm saline. Temperature. The preparation was kept warm on a metal top table with electric lamps and woollen cloths, by which means its temperature, recorded to f' C., was carefully and easily controlled. Blood-pressure. The blood-pressure was recorded at intervals by means of a mercury manometer, 2 p.c. aqueous sodium citrate solution being used as an intermediate fluid. For this latter purpose sodium bicarbonate solution was avoided, as it was discovered in an early experiment that, with a falling blood-pressure, the CO2 concentration of the blood was markedly altered by diffusion of the manometer fluid. Glucose infusion. In certain experiments an aqueous solution of glucose was infused. This was done by means of a constant infusion pump driven by an electric motor through a calibrated P a 1 m e r variable gear, by which means the glucose solution could be easily and accurately infused at a given rate per kg. of body weight. Air analysis. The preparation was allowed to breathe the room air and to expire into a specially light D o ugla s bag of 20 litres capacity, fitted directly over the expiratory valve by means of a rubber stopper. At intervals of 1 hour samples of expired air were collected during periods of half-hour duration, except occasionally late in the experiment, when the preparation exhibited hyperpncea and the collection period had in consequence to be shortened. The expired air samples were analysed immediately in duplicate with the Wishart-Haldane apparatus, being transferred directly from the bag to the burette of the apparatus after mixing. The room air, analysed at the beginning and end of experimental periods, showed no significant alteration in composition.

METABOLISM OF DECEREBRATE CAT. 393 Blood analysis. Arterial blood was withdrawn during each air sampling period. This was done bv means of a three-piece, all-glass syringe, having a measured and negligibly small nozzle capacity. All air from this nozzle space and from the needle was displaced by a saturated aqueous solution of sodium citrate, and the blood sample taken by passing the needle down to the lumen of the carotid artery through the wall of a closed rubber tube fitted to the cannula and containing 2 p.c. sodium citrate solution. The blood was allowed to flow into the syringe under ita own pressure. In earlier experiments, 1*75 c.c. of blood were taken at each sampling period, and in later experiments this volume was reduced to 1 c.c. The blood C02 content was determined with the Van Slyke-Neill manometric apparatus, using whole blood in duplicate samples of 0-2 c.c. measured over mercury. The blood lactic acid was determined by a modification of the method of Friedmann, Cotonio and Shaffer, 1 c.c. and, in later experiments, 0 3 c.c. of blood being used'. The blood sugar content was ascertained by the Hagedorn-Jensen method, using 0'15 c.c. of blood. The accuracy of the results using these quantities of blood was adequately checked. In a small number of experiments in which glucose was infused the haemoglobin percentage was determined at the beginning and end of the experimental period. RESULTS. 1. The decerebrate non-eviscerate preparation. Except in a few instances the non-eviscerate preparation showed a fairly steady metabolic rate for a period of 8 or 10 hours with hourly withdrawal of blood. As no aseptic precautions were taken in operating, the preparation did not live beyond the second day. The blood-pressure in almost every instance was practically constant for a period of 4-6 hours after decerebration. In a few instances it showed no serious fall during a post-decerebration period of 8-10 hours, and when blood withdrawal was reduced to a minimum, a high blood-pressure was maintained for 24 hours. No deductions have been made from results obtained with non-eviscerate preparations showing a blood-pressure of less than 70 mm. of mercury. The oxygen consumption per kg. body weight varied to some slight extent from animal to animal, but for cats weighing about 2 kg. when 1 Any error due to the presence of citrate in the blood is constant throughout the experiments.

394 I. A. ANDERSON AND OTHERS. the body temperature was maintained at 36.50 C. it had for the experimental period of 8-10 hours a fairly constant value, usually about 360c.c. per kg. per hour. Values in the vicinity of 400 c.c. per kg. per hour were frequently obtained when the temperature rose to 37-38. The R.Q. was steady at a value between 0 7 and 0'8. Temporary fluctuations were obtained with changes in body temperature and minute volume of expired air. The minute volume of expired air of cats of about 2 kg. had an average value of somewhat less than 190 c.c. per kg. body weight when the temperature was 360 to 370 C. p.m. 10 11 1200 100 2 0 3 0 400 5 6 700 8.0 9 I- L A I I -~~~~~~~~~~~~~~~ p.m. a.m. 1000 10 11 12 hours.id I PA Ie ^f 2 p~~~~~~~~~ Fig. 1. Experiment 46. * - -. r 385 Temp. [axiu& 36 'C. B.P. \ ~~~~mm. \ 70 Hg. 60 _ 432 0,cons. cc. per L347 kg. per hr, [0-9 R.Q. 0-73 - 349 Mi..vol. expired air c.c. 140 perkg. e 90 Blood lactic [ cid 25 mg.1loo.c, 47 Blood 128 COS vol. p.c,. The blood lactic and blood C02 concentrations changed but little with values in the vicinity of 30 to 60 mg. p.c. and 30 to 45 vol. p.c. respectively, the lactic acid concentration tending to rise progressively to the higher value, and C02 to fall to the lower. In four experiments in which the blood sugar concentration of the non-eviscerate preparation was estimated it was found to be high immediately following decerebration. In three it continued to rise and later fell, as has been observed by other workers. At no time did it return to the normal blood sugar level, the lowest value recorded being 253 mg. p.c. 9 hours after decerebration. The results of a typical experiment are shown in Fig. 1. 2. The decerebrate eviscerate preparation. The life of the decerebrate

METABOLISM OF DECEREBRATE CAT. eviscerate cat was found to be shorter than that of the non-eviscerate preparation. This is not to be attributed to loss of blood during the operation, which is negligible. It is due, we believe, mainly to the effect of virtual removal of the liver from the circulation. The earlier deterioration of the preparation is associated with a progressive increase in the lactic acid concentration and a fall in the CO2 concentration of the blood. Our belief that this increased lactic acid concentration is due to the removal of liver function is in agreement with the findings of Kilb orn, S oskin and Thomas [1928], Cori and Cori [1929] and others. From an examination of the lactic acid concentration of the blood immediately before and after evisceration, and in eviscerate preparations in which muscles have been dissected for the estimation of glycogen, it would appear that operative treatment per se may cause a considerable a.m. p.m. 11 1200 l 2 300 400 5 0 600 70 80 900 1000 hours lactic acid concentration of theblood._, 2 165. Blood [38 lacticic -cid 32 Blood 24 Vol. P.C. 1 7 LI ~~~~~~~~~~~93 FA mg.sloocr.a. ~~~~~~~~~~42 Fig. 2. Experiment 34. lactic acid production. Coincident with the operation of evisceration the lactic acid concentration of the blood showed a sudden rapid increase which was followed by a much slower increase (Fig. 2, Exp. 34). In order to have the blood lactic acid concentration as low as possible at the time of evisceration we have made a practice of allowing 14 hours to elapse between the operations of decerebration and evisceration. The rationale of this procedure is based on the grounds that the liver plays a part in the removal of the lactic acid which is produced by ether anaesthesia (Hinsey and Davenport, 1929) and the operation of decerebration. Even in the absence of any operative treatment other than the withdrawal of blood at the rate of 1-75 c.c. per hour, the blood of the decerebrate cat showed, after evisceration, a progressive increase in lactic acid and a proportionate fall in CO2 concentration, neither of which was shown to the same degree in the non-eviscerate preparation. In two experiments only was there any exception to this. In Exps. 39 and 42 a very slight fall in lactic acid concentration and a concurrent rise in C02 395

~~~ ~~~~~o f ~~~~~~~mm. 396 I. A. ANDERSON AND OTHERS. concentration occurred for a short period following evisceration. This was followed by a progressive lactic acid increase and C02 fall. The rise in blood lactic acid concentration following evisceration did not occur at the same rate in all preparations. In some instances it was very rapid (Fig. 5, Exp. 31). Usually, following the initial rapid rise coincident with the operation, successive blood samples showed a rise of 7-8 mg. p.c. per hour with a concurrent proportional fall in blood C02 concentration. Roughly, the latter fell about 1 vol. p.c. for each 6 mg. p.c. increase in lactic acid. This slower rise continued until the blood lactic a.m. p.m. goo' 10 11 12 1 co 2 3 400 5 6 7 8 90 38 Temp. faxilla] 140 B.P. Hg. c~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~cc ipere / \ 4153 095 kg.per h. Min.vol.PC _ [ ~ ~~~~~~~~~~~~ lactic 27 mg./l OOc.c. :o t. [ 2 C45 op Fig. 3. Expperiment 33. acid reached a concentration of about 80 mg. p.c., the blood 002 being then about 25-30 vol. p.c., after which, as a rule, the rise of lactic acid and the fall of 00 2 content of the blood became much more rapid (Fig. 2). Up to this "break point," which occurred within 5 hours of evisceration, the oxrygen consumption, the minute volume of exrpired air and the R.Q., as a rule, showed no very rapid changes, after discounting any temporary disturbance in these values immediately following the operation. Beyond this point they were no longer steady. Almost without exrception, associated with a rapidly rising blood lactic acid concentration and falling blood 002 concentration, the minute volume of exrpired air, oxygen consumption and R.Q. became greatly heightened (Fig. 3, Exrp. 33).

METABOLISM OF DECEREBRATE CAT. 397 The blood-pressure, although not affected immediately by evisceration, showed a greater tendency to fall than in the non-eviscerate preparation. This fall was, however, not severe until the "break point" was reached, when it became much more rapid. The blood-pressure in the eviscerate preparations was as a rule between 90 and 100 mm., and in one or two instances even 120 to 140 mm., of mercury at the "break point." Prior to the "break point" blood-pressures below 80 mm. of mercury have not occurred, and records after the "break point" have been stopped when a blood-pressure of 40 mm. of mercury was reached. When glucose was administered, a 4 p.c. solution was infused usually at the rate of 2 c.c. per kg. body weight per hour plus a volume equal to the volume of blood withdrawn (1 c.c. or 1-75 c.c. per hour). In Exp. 39, in which a 2*5 p.c. glucose solution was infused at the much higher rate of 5*2 c.c. per kg. per hour, the blood-pressure rose somewhat. The oxygen consumption per hour for cats weighing about 2 kg. at a body temperature of 36.50 C. had an average value in the vicinity of 245 c.c. per kg. of the eviscerated preparation. This value varied considerably with changes in body temperature and pulmonary ventilation and an average taken from a larger number of experiments, irrespective of body weight and temperature, is approximately 270 c.c. per kg. of eviscerate preparation. In the more stable preparations the oxygen consumption usually approximated the lower value. For instance, in Exp. 42 for a post-evisceration period of 6 hours with hourly analysis, maximum and minimum values of oxygen consumption per kg..per hour of 269 c.c. and 249 c.c. respectively were obtained. Here the blood lactic acid concentration had risen to only 76 mg. p.c. 5j hours after evisceration. Beyond the " break point " the oxidation rate greatly increased, rising in some instances to over 400 c.c. per kg. of eviscerated preparation per hour. That this terminal rise in oxygen consumption was due to increased movements of respiration is suggested by the similarity of curves of 02 consumption and minute volume of expired air. In the instance of Exp. 39 (Fig. 4) these values ran almost exactlv parallel. From our figures for the oxygen consumption of a 2 kg. cat with body temperature of 36.50 C., we have calculated the oxygen consumed per hour by 1 kg. of muscle and of abdominal viscera respectively, assuming that the oxidation rate of the muscles was not altered by the removal of the viscera, and attributing to muscle any oxygen consumed by the remaining tissues other than muscle. For this purpose we have taken the combined weight of the liver, spleen, pancreas, stomach and intestines as 10 p.c. of

398 I. A. ANDERSON AND OTHERS. the total weight of the cat, a figuire which our experimental data show to be approximately correct. The muscles we have taken as constituting 44 p.c. of the total weight, a figure given by Ferguson, Irving and Plewes [1929]. The figures are given in Table I. a.m. p.m. 9go 10 1100 12 1 2 3 4 5 6 7 8 9 hours 36 Temp. (axilla C. 10, Weigh 067 cfc. per kg. per hr. O'76.~~~~~~~~~~~~~~~~~9 g./loc H Min. vol.w expired viscera evisceration c.c. perhr.oacons. ar c.c. 2 per kg. Blood lactic acid 0- ~~~~~~~~~~~38 [34 Blood CO. oontent 18 Volo P.C. 308 Blood -164 [ mg./liooc.c. sugar Fig. 4. Experiment 39. TABLEm L Weight of cat 02 cons, after minus weight 02 cons, evisceration of abdominal before viscera eviscemation Cc. per hr. 02 cons. 02 cons. P.C. of total Weight removed, (c.c. per hr. c.c. per hr. perqk. of of muscles of viscera 02 cons. of cat including per kg. of per kg. of cat minus per kg. per kg. of due to (kg.) liver whole cat) whole cat viscera of muscle viscera viscera 2-0 1-8 360 220 245 500 1400 39 (Average of figures for cats of about 2 kg. with temperature of 36,50 C., expressed on 2 kg. basis.) The percentage of the total oxidation due to viscera on this basis is 39, which is in agreement with the figure given by Hunt and Bright [1926]. When the minute volume of expired air after evisceration per kg. of eviscerated cat was compared with that before evisceration per kg. of non-eviscerated cat, it showed no diminution, In this it differed from the oxygen consumption which was diminished by some 32 p.c. This is readily

METABOLISM OF DECEREBRATE CAT. explained on the grounds that the increased lactic acid production on evisceration, acting through the respiratory centre, maintained a high pulmonary ventilation while the oxidative metabolism per kg. was lowered. After the " break point" had been reached the minute volume rapidly increased and such values as 417, 504, 570, 636 and even 645 c.c. per kg. were observed. In this last instance the preparation weighing 2'92 kg. after evisceration was breathing at the rate of 44 respirations per minute with a tidal air of 42-8 c.c. The blood CO2 concentrations coincident with the minute volumes given above were 20, 18, between 23 and 31, 17 and 14 vol. p.c. respectively, while the R.Q.'s were 0 95, 0-87, 1.1, 0 99 and 0-86. Before the "break point" when minute volumes of 200 c.c. per kg. were observed the R.Q. in these preparations was 0-85, 0-801, 0 90 and 0-89 respectively. In this last instance the R.Q. was lower after the " break point" than before it. The blood CO2 concentration curve for this experiment (No. 36) offers a ready explanation of this. When the higher value, 0X89, was obtained the blood CO2 concentration was falling at the rapid rate of about 5-5 vol. p.c. per hour, while at the lower value of 0-86 the rate was only about 1*75 vol. p.c. per hour. The actual blood CO2 concentration in the latter instance had fallen to the low value of 14 vol. p.c., the lowest with one exception that we have observed. The R.Q. was less steady than with the non-eviscerate preparation. When the entire period following evisceration was considered the R.Q. varied between 0*76 and 10, and in one instance, Exp. 31, it reached 1x16. When the metabolism was steady the values recorded were fairly constant in the vicinity of 0*85 or less. A steady metabolism was indicated by a fairly constant oxygen consumption, and a rate of fall of blood CO2 concentration not greater than about 15 vol. p.c. per hour accompanied by a correspondingly slow rise in the minute volume of expired air. Higher and varying values Of R.Q. have been observed to occur mainly after the "break point." In some instances they also occurred immediately following evisceration. A marked rise in blood lactic acid and a fall in blood CO2 concentration were found to be associated with these higher quotients. In the main our results go to show that the R.Q. of the decerebrate eviscerate cat runs parallel with loss of blood CO2 which occurs with rise 1 In this experiment (Exp. 31, Fig. 5) the blood C02 concentration fell below the " break point" immediately following evisceration, accompanied by hyperpncea. The lowest minute volume recorded after evisceration was 390 c.c. per kg. which was associated with a respiratory quotient of 1*16. 399

400 I. A. ANDERSON AND OTHERS. in blood lactic acid. If the rise of lactic acid is rapid and the fall of C02 rapid the R.Q. continues to rise or remains high, but if the fall of C02 is slowed the R.Q. tends to fall from the high value to which it has been raised. It would appear that during the post-evisceration period prior to the "break point " the qikali reserve is capable of coping with the lactic acid production. During this period the blood C02 concentration is steadily p.m. 1 0 2 * 3 4 5 0 6 7 0 8 90 ho. 37 Temp. (axilfa] 36 Ic.. [ 40 Hg. [ 358 O, cons.. 0.0. per. 251 kg. per hr..- *! X - - \- ~~~~~~~~~~R.Q. I I -571 ~~~~~~~~~~~~~~~Min. vol. I expired air c.c.. [18 per kg. 15 Blood lactic ~ ~~ X CO, Fig. 5. Experiment 31. falling and in all probability this maintains the R.Q. at a higher level than would be shown by metabolism alone. As yet we have no accurate means of determining to what exatent the 002 displaced from the body fluids elevates the R.Q. of the eviscerate preparation above that due to its oxidative metabolism. This question of the validity of the apparent R.Q. of a muscle preparation was early criticized by Verz6,r [1910], and evidence that abnormal ratios were largely due to blowing off of 002 from the bo,dy fluids was put

METABOLISM OF DECEREBRATE CAT. forward by Kilborn [1928] and by Ferguson, Irving and Plewes [1929]. Corkill, Dale and Marks [1930], working on the spinal eviscerate cat, have put forward some evidence in support of the assumption that the R.Q. of this preparation is unity, or about unity. Although we recognize how greatly the conditions of our experiments differ from theirs we feel, in the light of our results, that the figure which they quote for the respiratory quotient is affected by causes other than the oxidative metabolism of the preparation. We have found consistently in our experiments that when the blood C02 concentration falls below a value of 25-30 vol. p.c. the decerebrate eviscerate preparation is in a state of fairly rapid deterioration. Analyses continued for some 4 hours or longer after this point have shown a falling blood-pressure, unstable and, for a time, abnormally high values for minute volume of expired air, oxygen consumption and R.Q., and ultimately a rapidly falling oxygen consumption. Corkill, Dale and Marks point out that six out of nine of their experiments show at some stage of the experiment a R.Q. of unity or very near unity, and it seems to us significant that of these six experiments not one has a blood plasma C02 capacity above 30 vol. p.c., and of the fifteen blood plasma C02 capacity values recorded for these experiments, thirteen are below 25 vol. p.c. On the other hand, of the three experiments with lower R.Q. values ten blood C02 values are recorded, six of which are above 30 vol. p.c. and only three below 25 vol. p.c. These figures seem all the more significant when consideration is taken of the fact that blood plasma C02 capacity figures are normally higher than those for whole blood C02 content. Further, the values for oxygen consumption of the spinal eviscerate cat given by Corkill, Dale and Marks are lower than those we have obtained with the decerebrate eviscerate cat. This higher oxygen consumption of the decerebrate preparation is probably to be attributed to its respiratory movements, but it seems worthy of comment that the two lowest R.Q.'s recorded by these workers are associated with the two highest oxygen consumptions, viz.: O consumption per kg. weight Blood of whole plasma CO2 Exp. no. R.Q. animal per hr. capacity 8 084 230 Not recorded 9 087 205 36 In preparations in which glucose was infused we have obtained values of R.Q. and oxygen consumption which are in no way different from those 401

402 I. A. ANDERSON AND OTHERS. obtained in the other experiments. In Exp. 39 the blood sugar content rose from 164 to 300 mg. p.c. during 9 hours after evisceration. The R.Q. remained at a value of from 0-81 to 0*82 over a period of 4 hours following evisceration when it began to rise with an increase in the pulmonary ventilation, the blood C02 concentration having fallen to 30 vol. p.c. SUMMARY. The changes in the carbon dioxide and lactic acid concentrations of the blood, and in blood-pressure, pulmonary ventilation, oxygen consumption and R.Q. of the decerebrate cat caused by evisceration are recorded and briefly discussed. The condition of the eviscerate preparation is correlated with changes in the carbon dioxide and lactic acid concentrations of the blood, and its R.Q. is observed to run parallel with the loss of blood carbon dioxide which occurs with rise in the lactic acid concentration. Respiratory quotients of 0*85 or less are obtained. Higher values are associated with lowering of the carbon dioxide content of the blood. If the rise in lactic acid and fall in carbon dioxide concentration are both rapid the R.Q. remains high, but whenever the fall in carbon dioxide concentration becomes slowed, although the lactic acid concentration may continue to rise, the R.Q. tends to fall from the high value to which it has been raised. We wish to express our thanks to Prof. T. Graham Brown, F.R.S., for guidance in the handling of the decerebrate preparation. Part of the expenses of the work was defrayed by a grant from the Medical Research Council whom we wish to thank. REFERENCES. Cori, C. F. and Cori, G. T. (1929). J. Biol. Chem. 81, 387. Corkill, A. B., Dale, H. H. and Marks, H. P. (1930). J. Physiol. 70, 86. Ferguson, J. K. W., Irving, L. and Plewes, F. B. (1929). J. Phy8iol. 68, 265. Hinsey, J. C. and Davenport, H. A. (1929). Amer. J. Phy8iol. 88, 286. Hunt, H. B. and Bright, E. M. (1926). Amer. J. Physiol. 77, 353. Kilborn, L. G. (1928). J. Physiol. 66, 403. Kilborn, L. G., Soskin, S. and Thomas, J. C. (1928). Proc. Roy. Soc. Can. 22, Sect. 5, 169. Verzdr, F. (1910). Bsochem. J. 34, 61.