has reported the results of a similar study. Roche) was injected intravenously before bleeding, and J.U.

Transcription

1 PULMONARY VASOMOTOR RESPONSES OF ISOLATED PER- FUSED CAT LUNGS TO ANOXIA AND HYPERCAPNIA. By HELEN N. DUKE.' From the Department of Physiology, University of Edinburgh. (Received for publication 13th June 1950.) RECENT observations in the intact animal indicate that a decrease in 02 concentration in the inspired air may act, as does CO2, on the pulmonary vessels to produce constriction [for references see Duke, 1949 a; Dirken and Heemstra, 1949 a, b, c; Nisell, 1950]. Isolated perfused lungs provide a means of determining whether these effects are independent of neuro-humoral reflexes. When this investigation was begun, Lohr [1924] had used this technique to study pulmonary vasomotor responses of the cat in response to anoxia and hypercapnia. The same problem had been investigated in anaesthetized cats [Retzlaff, 1913; Wearn, Ernstene, Bromer, Barr, German and Zschiesche, 1934; Von Euler and Liljestrand, 1946; Logaras, 1947] and in the cat heart-lung preparation [Drinker, Churchill and Ferry, 1926]. The results considered in the present paper dealing with the effects of 02 and CO2 on the isolated lungs of this species were briefly reported [Duke, 1949 b, 1950] earlier. Since this investigation was begun, Nisell [1948, 1950] has reported the results of a similar study. METHODS. Isolated cat lungs were set up and perfused using essentially the same technique as that described by Daly [1938] for the lungs of dogs. Cats weighing more than 2-0 kg. were anaesthetized with intraperitoneal pentobarbitone (0.03 g./kg.) or chloralose (0.1 g./kg.) and bled from the carotid artery until death occurred. Heparin (1000 I.U. "Liquemin," Roche) was injected intravenously before bleeding, and J.U. were added subsequently to each 100 c.c. blood collected. Artificial ventilation (Starling "Ideal" pump) was maintained during bleeding and subsequent dissection. Cannulw were inserted into the trachea, the left auricle and the pulmonary artery, and the ventricles were compressed by tying them tightly with tape. When positive pressure ventilation was used during perfusion the lungs and heart were left in situ, but when negative pressure ventilation was used they were removed from the chest. I Now at the Royal Free Hospital School of Medicine, London. 75

2 76 I)uke The majority of experiments were performed under negative pressure ventilation. For this purpose the lungs were supported on a perspex plate and enclosed in a 9-litre glass jar, which was placed on its side on a warmed operating table. The open end of the jar was closed by a wooden lid through which tubes passed to the pulmonary artery, left auricle and trachea. The pressure in the chamber was reduced by means of a Starling "Ideal" pump arranged to remove c.c. of air out of the chamber at each cycle. The negative pressure in the chamber (read on a water manometer) was varied at will by adjusting a screw clip on a rubber tube leading into the chamber from outside. In the majority of the experiments the pressure was made to oscillate rhythmically between - 1 and - 10 cm. H20 at a rate varying between 12 and 16/minute. Approximately 500 c.c. of warm saline were placed in the chamber before perfusion was begun, to keep the atmosphere moist. The trachea was usually connected to a T-piece containing Siebe-Gorman inspiratory and expiratory valves. A small recording spirometer could be attached at will to the expiratory side, so that a record of the tidal air could be obtained for 3 to 6 respirations when desired. When positive pressure ventilation was used, the lungs were inflated by a Starling "Ideal" pump, and the tidal air was usually recorded under a constant positive pressure of 5-12 cm. H20 by the method of Konzett and Rossler [1940], with the omission of soda lime in the circuit. Perfusion was performed at constant volume inflow with a Dale- Schuster pump, using the animal's own heparinized blood (at 360 C C.). The capacity of the system was 40 c.c., excluding the venous reservoir (which contained c.c. blood at the beginning of perfusion) and the pulmonary vessels. The pulmonary arterial pressure and venous reservoir blood-volume were recorded as described in a previous paper [Duke, 1949 a]. The latter indicates inverse changes of lung bloodvolume [Daly, 1928]. The gas mixtures used were from commercial cylinders, and stored in Douglas bags. The Douglas bags were attached to the input of the Starling pump or to the inspiratory side of the valves, in the positive or negative pressure experiments respectively. The capacity of the apparatus from the point of attachment of the bag to the bifurcation of the trachea was approximately 100 c.c. in the negative pressure experiments and 150 c.c. in the positive pressure experiments. Neon, or hydrogen, was stored in 3-litre rubber anaesthetic bags. These were attached directly to the tracheal cannula during negative pressure ventilation of the lungs, and rebreathing of the gas from the bag was allowed for short (2 min.) periods. Analysis of 02 and CO2 in samples of blood from the pulmonary arterial tubing was by the method of Peters and Van Slyke [1932].

3 Pulmonary Vasomotor Responses of Perfused Cat Lungs Hsemoglobin was estimated as cyanhaemoglobin [Peters and Van Slyke, 1932], using a Hilger Spekker photometer and a Chance No. 5 filter. The percentage blood 02 saturation was calculated approximately from the Hb. concentration and 02 content without correcting for dissolved 02- RESULTS. Before making any tests it was necessary to show that cats' lungs could be perfused for a suitable experimental period without gross pathological changes. Contrary to previous experience with the cat heart-lung preparation perfused with defibrinated blood or blood with added Chicago blue [Knowlton and Starling, 1912; Newton, 1932], or isolated cat lungs perfused with diluted defibrinated blood [Builbring and Whitteridge, 1945], it was found that, with the method described, perfusion could be continued for over 5 hours without onset of gross cedema. Pulmonary Arterial Pressure, Lung Blood- Volume and Tidal Air Changes during Perfusion The pulmonary arterial pressure was often as high as cm. of blood, with a pulmonary arterial inflow of only 50 c.c./min. during the first few minutes of perfusion. The pressure tended to fall with continued perfusion and ventilation, but the rate of decrease of pressure was often hastened by one or two extra positive pressure inflations of the lungs, especially if these were performed with expired air. The pulmonary arterial inflow was gradually increased to c.c./min. as the pressure fell. After the first 10 to 15 minutes of perfusion the pulmonary arterial pressure and lung blood-volume showed little change for 2-3 hours at constant ventilation and perfusion. The tidal air was initially c.c. but tended to decrease during the course of the experiment. Pathological Changes during Perfusion. At the end of each experiment the lungs were examined externally and after slicing. The main pathological findings were cedema, congestion and atelectasis, most prominent in the lower lobes and the under surfaces of the lungs generally. In 35/48 experiments the lungs showed only slight lesions in one or two lobes, and in only two cases was cedema so gross as to be apparent in the larger airways. Histological sections from the lobes which had appeared best and worst on macroscopical examination confirmed the presence of congestion and cedema, but also showed that ample functioning lung tissue was always present. Another indication of the absence of pulmonary cedema was the relatively slight VOL. XXXVI, NO

4 78 Duke changes in blood Rb concentration during the first 3 to 4 hours of perfusion (see fig. 1). Hb. gm ExptNIos 0o 8 Xx i X 13 * a- 4D X-.-'0X X a, 9Q I I - -x a- - touts Oft perfusion FIG. 1.-Blood hemoglobin concentration c,uring the course of perfusion in seven separate experiments. THE EFFECT OF VARIATIONS IN THE VENTILATING GAS MIXTURE. A. Carbon Dioxide. If the concentration of C02 inhaled was increased from the small amount present in atmospheric air to 5 or 10 per cent., an increase of pulmonary arterial pressure was produced. The pressure rose after a latent period of seconds to reach a maximum in 3-6 minutes, thereafter it often fell slightly, but was maintained above the control level until air was reventilated. Table I shows that 10 per cent. C02 TABLE I.-CHANGE FROM AIR TO AIR CONTAINING TimneDrto u I i i 10 PER CENT. CO2. Expt. from Drto begir g of C2 P.A.p. Change. L.B.V. Blood C02 of per- i., Change, volume fusion. stration. Actual Per c.c. per cent. cm. cent.. hr. min. min. blood. increase. Before. After * * P.A.p. = mean puhlmonary arterial pressure. L.B.V. = lung blood-volume. Blood 0 volume per cent. Before. After. 16*77 16* *31 10*

5 Pulmonary Vasomotor Responses of Perfused Cat Lungs usually increased the pulmonary arterial pressure from 10 to 12 per cent. above its original level, although the response was sometimes greater (up to 96 per cent.). Fig. 2 shows a response to 10 per cent. CO2. In only 2 out of 44 preparations did CO2 fail to produce an increase of pulmonary arterial pressure, and these preparations were also less than normally reactive to adrenaline and other stimuli. 79 A B FIG. 2.-Expt. 13. Cat <D 3-17 kg. Pentobarbitone anmesthesia. Perfusion begun a.m. Negative pressure ventilation 0 to -10 cm. H20. (a) 2.53 p.m. Ventilating gas mixture changed from air to 10 per cent. C02 in air. (b) 2.59 p.m. Still respiring 10 per cent. C02 in air. Pulmonary arterial pressure is maintained at a new level. Slight increase of volume of blood in venous reservoir. V.R. =venous reservoir. P.A.p. =pulmonary arterial pressure. The lung blood-volume was unaltered or slightly decreased during CO2 inhalation (see Table I). The apparent increase in lung bloodvolume seen during the early stages of C02 administration in fig, 2 is due to the change in the volume of blood in the recording apparatus consequent upon an increase of pulmonary arterial pressure. No change in lung blood-volume could be detected in tests with 10 per cent. CO2 when the apparatus error had been minimized by clipping

6 80 Duke off the tambour and reading the pressure changes on the manometer only Ṫhe pressor response to CO2 appeared not to have a mechanical origin in bronchomotor changes. Although C02 apparently produced bronchodilatation in some preparations (see fig. 3) this was not constantly present and, furthermore, the bronchomotor response did not coincide in time with the pressor response, nor did the magnitude of the two responses run parallel. A B C Fmc. 3.-Expt. 33. Cat ~ 2-5 kg. Chloralose aniestliesia. Perfuision begunii Ilk 1. rei.. Respiration pump delivery,50 c.c/stroke. Positive pressure ventilation at 5 cm. H20. (A) 11I.28 a.m. Frorn air to 10 per cent. CO2 in air at signal. (B) p.m. 10 per cent. CO02 in air ventilated between signals. (C) 1.32 p.m. 10 pcr cent. CO2 in air vcntilated between signals. T.A. -tidal air. a decrease in the excursion of the lever denotes a decrease in resistance to inflationl. Ventilation of isolated lungs with air rapidly decreased the amount ofc2in the blood. At the enm1 of j--1 hour the pulmionary arterial blood somnetimies contained as little as 3-3 vol. per cent. (902; this was increasedto approximately 20 vol. per cent. after 6-10 minutes inhalation of 10 per cenit. ( 02 in air or N2 (see Table 1). The bloodo02 conicentration was also decreased by the addition of 10 per cent. (902 to the respired air, partly because of the shift in the 02 dissociation curve caused by (902, and partly because theo02 tensioni of the respired gas mixture was lowered as the result of addition of (90., However, the pressor response, to (902 appeared to be inidepenident of the blood and the alveolar 02 content, because ventilation with 5 10 per cent. (902 in air, N2 or 0,

7 Pulmonary Vasomotor Responses of Perfused Cat Lungs 81 produced an increase of pulmonary arterial pressure if the gas mixture respired previously had been air, N2 or 02 respectively. In 3 experiments the blood was exposed to an atmosphere of 10 per cent. CO2 in 02 or N2 before it was pumped into the lungs. The pulmonary arterial blood in these preparations had a more normal CO2 content than in preparations in which such a procedure was not adopted. It was found that, under these conditions, an increase in the CO2 content of the ventilated gas also caused an increase of pulmonary arterial pressure. B. Oxygen and Oxygen Lack. Ventilation of the lungs with 02 or 5 per cent. CO2 in 02 instead of air or 5 per cent. CO2 in air, for 3-16 minutes did not produce any constant effect on the lung blood-vessels detectable under the conditions of these experiments. Table II shows that the 02 saturation of the pulmonary arterial blood, which was per cent. during ventilation with air, was increased to per cent. as a result of the tests with 02. The negative results could not therefore have been due to a failure of 02 to reach the alveoli. Timne TABLE II.-CHANGE FROM AIR TO 02. Duration Expt. begoni of 02 No. beginning Admuin- P.A.p. Change. Blood 02 of per-.. volume fusion. stration. Actual Per per cent. cm. cent. hr. min. min. blood. increase. Before. After B * nil ll B -12* *9 Blood CO, Per cent. volume Per cen. per cent. Saturation 03. Before. After. Before. After * *0 99* Inhalation of N2 or 5 per cent. CO2 in N2, instead of air or 5 per cent. CO2 in air, for 3-10 minutes caused an increase of pulmonary arterial pressure after a latent period of 30 seconds to 2 minutes. The pressure continued to rise throughout the whole period of ventilation with N2 and returned to its original level when the gas mixture was changed to air. The pressor response was unaccompanied by marked changes in lung blood-volume and was independent of tidal air changes. The magnitude and the latent period of the response to N2 varied in different preparations and in the same preparation at different times. There was sometimes no vasomotor response to N2 during the first hour of perfusion, or if a pressor effect was present at this time it was usually smaller than later in the course of the experiment. In other

8 82 Duke preparations the effect was unchanged for 2 to 3 hours or it diminished. The variation in the size of the response did not appear to be correlated with the changes in blood 02 or CO2 (Table III), or with the total length of time for which N2 had been inhaled before each test. TABLE III.-CHANGE FROM AIR TO N2. Time DUratiOn Expt. from OfNu No. beginning Adnin P.A.p. Change. Blood 02 Blood CO, of perfusion. stration. volume volume Per cent. saturaton Actual Per per cent. per cent. Cm. Cent. hr. min. mm. blood in1crease. Before. After. Before. After. Before. After * ] * * * I * * * nil * Left auricular sample. Tests were made to see if the pulmonary pressor response to nitrogen was a specific effect, or due to the reduction of alveolar 02 concentration. The percentage increase in pulmonary arterial pressure in response to the inhalation of gas mixtures containing various amounts of 02 in N2 was measured. As long as these tests were completed within approximately i hour they were not vitiated by changes in the sensitivity of the preparation. It was found (see figs. 4 and 5) that the percentage increase of pressure was greater in the tests in which the gas mixture contained a lower proportion of 02. An attempt was made to determine the degree of sensitivity of the pulmonary blood-vessels to 02 lack by determining the least reduction in 02 content of the inspired air which was sufficient to cause a pulmonary pressor response. It was found that the response occurred constantly with N2 containing 10 per cent. or less 2, and variably with N2 containing 15 per cent. 02. The effects of N2 were also compared with those of neon or H2. These latter gases produced pulmonary pressor responses as large as those produced by N2 when given in the same preparation and under the same conditions of test (see figs. 6 and 7).

9 Pulnionary Vasoitnotor Responses of PerfuLsed (Cat Lungs 3,S 3 Expt. Po. * O i00 I'm. 4. The effe(t of ventilation of the liungs with mixtures of O., anid N. for 3- t pe.rio(ds. Ordinate =percentage increase of pulhnonar-t arterial pressorc albox-e its iiitial clue. Abscissa =0, tension of inspired air in mmiin. Hg. Thliree separate experimilents are show a. A B C Fio. 5.-Expt. 42. Cat 3-2 kg. Chloralose anwsthesia. Conistant positive pressure ventilation at 8 cmi. H.,0. Top tracing-tidal air. Respiratory pumip dlelivery -50 c.c./stroke. Perfusion begun amn. Ventilation withi air. Test gases inhaled during signal. (A) 2.41 p.m. 10 per cett. 02 i N2. (B) 2.50 p.m. 5 per ceoit. 02 i' 'V (C) 3.02 p.m. Pore N_.

10 84 Duke All tests with 02, CO2 or N2 were carried out under negative or positive pressure ventilation and in preparations in which chloralose or pentobarbitone had been used as the anaesthetic. No difference in results was obtained which could be attributed to these variables. A B C D FiG. 6. Expt. 43. Cat $ 3.75 kg. Pentobarbitone anmesthesia. Perfusion begun p.m. Blood flow 150 c.c./min. Negative pressture ventilation 0 to -10 cm. H20. Ventilation with air. Test gases inhaled during signal. (A) 2.14 p.in. N2. (C) 2.39k p.m. Neon. (B) 2.22j p.n. 02. (D) 2.55 p.m. N2 At 2.49 p.m. a test witrh 02 gave negative results. A B C FIG. 7. Same experiinent as fig. 6. (A) 5.7 p.m. H2. (B) 5-27 p.m. 02. (C) 5.35 p.m. N2. Control tests were also made of the method of administration of the gas mixtures, using Douglas bags filled with air instead of a test gas mixture. These tests were without effect on the pulmonary arterial pressure. Dihydroergotamine (D.H.E. 45, Sandoz. conc. 1: 5000) or atropine sulphate (conc. 1: 250,000) added to the blood either separately or together had no influence on the pulmonary vascular responses described here. Dihydroergotamine, in the concentration used, reversed or inhibited pulmonary pressor responses to adrenaline.

11 almonary Vasomotor Responses of Perfused Cat Lungs 85 DISCUSSION. During constant volume inflow perfusion of isolated cat lungs through the pulmonary artery, ventilation of gas mixtures containing 5-10 per cent. C02, or N2 containing less than 15 per cent. 02, produces an increase of pulmonary arterial pressure. This response is probably the result of active pulmonary vasoconstriction since it occurs under constant conditions of circulation and ventilation, although further work must be done to exclude the participation of the bronchial blood-vessels. Inhalation of pure neon and pure H2 produces a pressor response which is similar to that produced by pure N2, so that it is probable that the effects of N2 are due to anoxia. Since atropine and dihydroergotamine do not affect the pulmonary vasomotor responses to CO2 or anoxia, it is unlikely that adrenergic or cholinergic nervous elements are involved, unless there are such elements in the lungs, capable of remaining active for 5 hours of perfusion, but either unaffected by the drugs used or inaccessible by the route of injection. The available evidence is thus in favour of the change in alveolar air composition exerting a direct effect on some part of the pulmonary vascular bed. Other possibilities must also be considered, for instance the release of active organic or inorganic substances from the lung tissue. Inhalation of CO2 produces changes in blood ph which might be of sufficient magnitude to account for the pulmonary vasomotor response to this gas [Nisell, 1950; Duke and Killick, 1950], but the changes in blood ph during anoxia are smaller and less constant. Liberation of histamine during anoxia and hypercapnia seems to be excluded by the observation [Nisell, 1950] that the pulmonary vasomotor responses are not abolished by antergan. Further experiments [Duke and Killick, 1950] indicate that the pressor response to anoxia is the result of metabolic changes, possibly in the smooth muscle of the pulmonary vascular bed. These results will be published in detail shortly. The results described in this paper are in agreement with those of Nisell [1950] in so far as the pulmonary vasomotor responses to C02 and anoxia are concerned. Bronchomotor responses to 02 lack were not observed in the present series; this is possibly because the bronchi were not artificially constricted, as in Nisell's experiments, before the tests. Bronchial dilatation was, however, obtained during excess C02 inhalation. Hamilton [1940] and Cournand [1947] suggest that results obtained on the isolated lung preparation may not be applicable to the whole animal because the conditions of perfusion experiments are necessarily abnormal. Evidence has already been presented that there was no gross pulmonary pathological lesion in the present series. However, it must be admitted that there are disadvantages in using the present

12 86 Duke technique for problems connected with gas exchange, because the concentration of 02 and C02 in the pulmonary arterial blood is far outside normal limits. For this reason it is of particular interest that similar responses have been obtained in the intact animal. For instance, inhalation of gas mixtures containing only per cent. 02 has produced pulmonary vasoconstriction in man [Motley et al., 1947] and cat [Von Euler and Liljestrand, 1946; Logaras, 1947]; and excess C02 inhalation has produced pulmonary vasoconstriction in cat [Von Euler and Liljestrand, 1946; Logaras, 1947], mouse [Sj6strand, 1935] and dog [Hochrein and Keller, 1932]. Von Euler and Liljestrand's observation that the pulmonary arterial pressure is decreased during 02 inhalation may be correlated with the lower 02 saturation of the arterial blood in the whole animal. In the absence of detailed knowledge of the haemodynamic characteristics of the pulmonary vessels of the cat, it would be dangerous to speculate about the nature of the vessels participating in the responses from the character of the pressure and volume changes. It is likely that considerable species variations may exist in the pressure-volume relationship, because it has been shown [Duke, 1949 a, b) that the inhalation of C02 in the dog lung produces a predominant change in lung blood-volume with only a slight change in pressure. The present experiments show that the reverse obtains in the cat, and the cat lung appears to behave like that of the monkey towards C02 [Hebb and Nimmo-Smith, 1948]. It is not possible to assign a definite physiological role to the responses described in this paper. In the intact animal, cardiovascular and respiratory effects of anoxia and hypercapnia complicate the end result, and may counterbalance any direct effect on the pulmonary vessels. It remains to be discovered whether the change in alveolar air composition diminishes the lung blood-volume and thus redistributes blood in favour of the systemic circulation [as suggested by Hochrein and Keller, 1932], or whether the effect is to shut off unventilated areas of the lungs [Von Euler and Liljestrand, 1946]. It is also possible that cardiovascular and respiratory reflexes may be initiated from the pulmonary vessels under the conditions of anoxia and hypercapnia. SUMMARY. 1. A method for perfusing isolated cats' lungs with the animal's own heparinized blood is described, with some observations on the condition of the lungs during and after perfusion. 2. The pulmonary vasomotor responses to inhalation of various gas mixtures have been studied during perfusion of the pulmonary vessels at constant volume inflow.

13 Pulmonary Vasomotor Responses of Perfused Cat Lungs 3. Inhalation of gas mixtures containing 5-10 per cent. CO2 produces pulmonary vasoconstriction, as shown by an increase of pulmonary arterial pressure. 4. Inhalation of gas mixtures containing less than 15 per cent. 02 also produces a pressor response. 5. Ventilation of the lungs with pure 02 has no effect under the conditions of the experiments. 6. Ventilation of the lungs with pure neon or H2 produces similar effects to that produced by pure N2. 7. The pulmonary vasomotor responses to CO2 and 02 lack are not abolished by dihydroergotamine or atropine. ACKNOWLEDGMENT. Some of the expenses of this research were met by part of a block grant from the Earl of Moray Fund. 87 REFERENCES. BULBRING, E., and WHITTERIDGE, D. (1945). J. Physiol. 103, 477. COURNAND, A. (1947). Bull. N.Y. Acad. Med. 23, 27. DALY, I. DE BURGH (1928). J. Physiol. 65, 422. DALY, I. DE BuRGiH (1938). Quart. J. exp. Physiol. 28, 357. DIRKEN, M. N. J., and HEEMSTRA, H. (1949 a). Ibid. 34, 193. DIRKEN, M. N. J., and HEEMSTRA, H. (1949 b). Ibid. 34, 213. DIRKEN, M. N. J., and HEEMSTRA, H. (1949 c). Ibid. 34, 227. DRINKER, C. K., CHURCHILL, E. D., and FERRY, R. M. (1926). Amer. J. Physiol. 77, 590. DUKE, HELEN N. (1949 a). Quart. J. exp. Physiol. 35, 25. DUKE, HELEN N. (1949 b). J. Physiol. 108, 59P. DUKE, HELEN N. (1950). Ibid. 111, 17 P. DUKE, HELEN N., and KILLiCK, ESTHER M. (1950). Unpublished experiments. EULER, U. S. VON, and LILJESTRAND, G. (1946). Acta physiol. scand. 12, 301. HAMILTON, W. F. (1940). Trans. U.S. Amer. Ass. Adv. Sci. 13, 324. HEBB, CATHERINE O., and NIMMo-SMITH, R. H. (1948). Quart. J. exp. Physiol. 34, 159. HOCHREIN, M., and KELLER, C. J. (1932). Klin. Wschr. 11, KNOWLTON, F. P., and STARLING, E. H. (1912). J. Physiol. 45, 146. KONZETT, H., and ROSSLER, R. (1940). Arch. Exp. Path. Pharmak. 195, 71. LOGARAs, G. (1947). Acta physiol. scand. 14, 120. LOHR, H. (1924). Z. ges. exp. Med. 39, 67. MOTLEY, H. L., COURNAND, A., WERKO, L., HIMMELSTEIN, A., and DRESDALE, D. (1947). Amer. J. Physiol. 150, 315. NEWTON, W. H. (1932). J. Physiol. 75, 288. NISELL, 0. (1948). Acta physiol. scand. 16, 121.

14 88 Pulmonary Vasomotor Responses of Perfused Cat Lungs Nisi,LL, 0. (1950). Ibid. 21, Suppl. 73. PETERS, J. P., and VAN SLYKE, D. D. (1932). Quantitative Clinical Chemistry, vol. 2 (Methods). Baltimore. RETZTLAFF, K. (1913). Z. exp. Path. Therap. 14, 391. SJ68TRAND, T. (1935). Skand. Arch. Phy8iol., Suppl. 71. WEARN, J. T., ERNSTENE, A. C., BROMER, A. W., BARR, J. S., GERMAN, W. J., and ZSCHIESCHE, L. J. (1934). Amer. J. Physiol. 109, 236.