THE DEVELOPMENT OF AN INCREASED PULMONARY VASCULAR RESISTANCE BY LOCAL HYPOXIA. By H. HEEMSTRA. From the Physiological Institute, University of Groningen, Netherlands. (Received for publication 2nd December 1953.) IN rabbits in which the ventilation of each lung is separately controlled, unilateral breathing of a low oxygen mixture increases the vascular resistance of the hypoxic lung [Dirken and Heemstra, 1948 a, b]. The response develops slowly and reaches a maximum only after 8 hrs., when with severe hypoxia the fraction of the total pulmonary flow carried by the treated lung may only be about one-half the value originally recorded. Other workers have reported similar responses to hypoxia, but the description they have given indicates that the reaction is a more rapid one than we have observed. Thus in isolated perfused cat lungs in which reduction of the oxygen concentration of the ventilating gas mixture produces vasoconstriction [Nisell, 1950; Duke, 1951], the increase in resistance begins after a latent period of only about 30 sec. and continues for 4 to 10 min., after which the curve begins to flatten out. A similar reaction of the pulmonary blood vessels demonstrated by Atwell, Hickam, Pryor and Page [1951], Peters and Roos [1952], and by Rahn and Bahnson [1953] in anesthetized dogs also develops rather promptly, within 10 to 20 min. Although these results are in apparent disagreement with our own the data are not in fact comparable, since in our experiments the initial phase of the response has not been studied in detail, while in the experiments of the other authors quoted it is the initial intensity rather than the duration of the response which has been examined. It seemed desirable therefore to repeat some of our earlier experiments and to make the additional observations necessary to describe the initial effects of hypoxia on the rabbit lung in detail. METHODS. In most cases the rabbits were anesthetized with 1.5 g. urethane per kg. subcutaneously, but pernocton was used as an anaesthetic in some of the earlier experiments. Care was taken to maintain the rectal temperature at about 370 C. during experiment, since at very low body 83
84 Heemstra temperatures (310C.) the reaction is slowed down. Experiments on albino rabbits and on animals found to be pregnant at autopsy were discarded, since in some ways their responses to hypoxia are irregular. The technique for the determination of blood flow ratio in the right and left lung has been described by Dirken and Heemstra (1948 b) as the " spirographic method ". The method depends on the principle that the ratio of oxygen uptakes, determined simultaneously for the left and right lung, equals the ratio of blood flow for the two lungs, when the pulmonary venous oxygen content is the same on both sides. This condition was fulfilled by having both lungs breathe pure oxygen from the closed circuits during the determinations, so that full oxygen saturation was attained independently of small differences in the alveolar oxygen concentration of each lung. It should be realized that by this method it is only possible to estimate the blood flow through one lung as a fraction of the total pulmonary flow, and that absolute values cannot be determined. It is possible that the method of recording led to an underestimate of the vascular reaction to hypoxia, since some relaxation of the vessels may have occurred during the brief period of exposure to a higher oxygen tension. However, for responses after 1 hr. this is unlikely to have been a significant source of error, since it has been shown [see Dirken and Heemstra, 1948 b, Table VIII] that recovery of the vessels after long exposure to hypoxia is slow and proceeds exponentially, with a time constant of about 1 hrs. This means that after 2 min. of ventilation with air only about 2 per cent recovery would have occurred. It might have been faster with oxygen but would still not materially affect the result. On the other hand, with exposures of less than 1 hr., the time course of recovery is not known and estimates of the vascular responses within this period may thus be significantly below the true values. At the start of an experiment the blood-flow ratio was determined twice in succession and the mean value taken as the normal control value. At zero-time one lung was switched from air to a gas mixture containing about 5 per cent oxygen and the other from air to a mixture containing about 60 per cent oxygen. Determinations of the blood-flow ratio were then made at 5, 15, 35, 60 min. and thereafter at intervals of an hour for eight hours. Then the lungs were again switched to air and the recovery process recorded hourly for a three-hour period. Unilateral sympathectomy was performed in some of the experiments by removing the upper part of the sympathetic chain, including the inferior cervical and the stellate ganglion, via the upper thoracic aperture. This approach had the advantage that it left the chest and pleura intact, and did not interfere with the respiratory movements so long as the phrenic nerve was not damaged. It is probable that with this procedure the major part of the sympathetic supply to the lungs was interrupted
Increased Pulmonary Vascular Resistance by Local Hypoxia 85 (T1, T2), but there may be some fibres from lower segments which bypass the stellate ganglion and, if so, they would have been left intact. RESULTS. In most of the experiments the right lung was used as the low oxygen lung. The fraction of total pulmonary blood flow, circulating normally through this lung, was found in 18 experiments to be between 0.505 and 0*609 (mean 0*555 and S.E.M. 0.0066). In each animal, the mean value of two successive determinations, obtained during breathing of air, was used as a normal control value, and is here referred to as the right or left "normal fraction" (F0). Subsequent values (F,), obtained during unilateral breathing of low oxygen in the same animal, were expressed as percentages of the normal fraction (100 F,/F,) and will be designated as the "relative flow-ratio ". The expression is useful not only as a means of following successive changes of flow-ratios in the same animal, but also as a means of comparing the responses of different animals. Table I summarizes the results of a series of experiments on 6 normal rabbits. TABLE I. Time. Experiment number: 3. 7. 8. 9. 15. 16. Mean. R. and L. air R. normal fraction, F0. 0 535 0 609 0.591 0.583 0.550 0*567 0*572 Unilateral hypoxia: R. inspiratory 02 per cent. 0 min. (L. ca. 60 per cent 02) 4-61 4*23 6-57 6-57 4.37 6-59 R. relative flow ratio per cent, 100 Ft/F0. 0,,,, 100 100 100 100 100 100 100 5 94.0 90*6 83-5 87-6 87.5 895 88*8 15,,,, 93.8 84.4 87-7 85.7 83 2 84-7 86*6 35,, 97.0 87 4 86-5 86*9 88*5 85.8 88-7 1 hr.,, 899 85 2 87 3 93 0 88*8 79-6 87.3 2 hrs.,, 85.2 81 6 840 85.7 82 7 70 0 81.5 3,, 83 0 87*9 82-7 81 7 67*1 68*2 78.4 4,, 77.8 70*5 76-3 81*9 49.8 58-3 69*1 5,,,, 80-9 69*5 78*8 73*1 465 585 67*9 6,,,, 78*7 69-0 70*7 68*2 44*5 52*7 64-0 7,,,, 72*5 65*1 69*7 69*5 44.9 51 9 62*3 8,,,, 72*7 62 8 66*9 59*6 46-7 51*9 60 1 8,, Recovery: R. and L. air 9,, 79.4 74.7 784 75*2 70*7 74.7 75.5 10, 83*9 82*2 81*7 85*1 82*1 86-0 83*5 11,,,, 86-3 85*4 85*3 89*7 92-7 91*5 88*5 At 5 and 15 min. the values were already significantly below 100 per cent, indicating an increased vascular resistance in the hypoxic lung. Between 35 min. and 1 hr. a partial return to the original blood-flow ratio could be observed, but then a slow progressive decrease began
86 Heemstra anew; and at 8 hrs. the flow was only 60 per cent of the normal fraction. It seemed, therefore, that there were two distinct phases in the progress of the reaction (fig. 1, A). The number of experiments forming the basis of the present report was not sufficient to determine statistically whether this description of %/ I 100 Rair. L60o%02. 90 R ca. 5%02. L 605 02. I I I R air. L air. 50 1 2 3 4 5 6 7 8 9 10 11 HOURS. FIG. 1.-Ordinate: blood-flow ratio as a percentage of the control value. A. Unilateral hypoxia and recovery. Mean of 6 experiments. B. Effect of 60 per cent oxygen in the contralateral lung. Mean of 4 experiments. C. Unilateral hypoxia (ca. 4 per cent 02) with homolateral (1) and crossed (2) sympathectomy. Mean of 3 experiments. the reaction was justified. Data from two other series of comparable experiments (unpublished work by Heemstra and Vriezen on 6 cortisoneand 6 ACTH-treated rabbits) were available, however, and the results of the three series have been analysed statistically. Kendall's [1948] method of m rankings (18 rankings of the 5-, 15-, 35- and 60-min. values)
Increased Pulmonary Vascular Resistance by Local Hypoxia 87 gave P < 0-025 for the hypothesis of random rankings. For these values the respective overall (column totals) ranking was 1, 4, 2, 3; in other words, the 15-min. value was on the average the lowest of the four values obtained during the first hour of unilateral low oxygen breathing. Moreover, the sign test indicated that the 15-min. value was less-p < 0-01-than the average of the 5- and 35-min. values, and that the 35-min. value was higher-p < 0-05-than the average of the 15- and 60-min. values (two-tail percentiles). The analysis thus shows that the response was, in fact, diphasic. A response of this kind might have occurred if, independently of the reaction of the hypoxic lung, some additional change was produced in the oxygen lung. To exclude this possibility four experiments were done with the right lung breathing air while the left one breathed the usual mixture of 60 per cent oxygen. TABLE II.-EFFECT OF HIGH OXYGEN. Time. Experiment number: 19. 20. 21. 22. Mean. R. and L. air R. normal fraction, Fo. 0*540 0-645 0-562 0-619 0-591 0 min. R. air, L. ca. 60 per cent 02 R. relative flow ratio per cent, 100 Ft/F0. 0,,,, 100 100 100 100 100 5,, 99 5 102-5 95-1 99-8 99-2 15,,,, 1009 101-7 96-0 100-2 997 35,,, 99 5 102-1 90 9 96-9 97 4 60,,,, 99.1 99.3 92-3 102-2 98-2 The results given in Table II show that the mean values (fig. 1, B) did not deviate significantly from 100 per cent. The high oxygen mixture caused if anything only a very slight shift of blood flow towards the oxygen lung. The diphasic character of the responses to hypoxia could not therefore be explained by the action of high concentrations of oxygen. The virtual absence of oxygen effects had already been observed earlier [Dirken and Heemstra, 1948 a] over much longer periods, but no special attention had then been given to the events of the first hour. The response to hypoxia was studied further in 15 tests on 8 rabbits in which unilateral removal of the stellate and inferior cervical ganglion was performed. This procedure did not significantly affect the response of the lung on the operated side when compared with that of the other lung (see fig. 1, C (1) and (2)); thus no evidence of the participation of the sympathetic nervous system in the response was obtained. Determination of Oxygen Saturation and Oxygen Tension of Mixed Arterial Blood. Changes in the oxygen content of the mixed arterial blood during unilateral hypoxia should be predictable from the data obtained with the spirographic method. Thus it would be expected that after an initial drop in the blood 02 at the beginning of the hypoxic period a rise
88 Heemstra which should be diphasic in character would follow. Re-examination of earlier results confirmed this expectation. In this work [Heemstra, 1948; Dirken and Heemstra, 1948 a], oscillations of oxygen saturation %HbO2 o MAXIMA o MINIMA 1 2 3 FIG. 2.-Mixed arterial oxygen saturation during unilateral hypoxia. HOURS during the first hour had been observed, but at that time they were thought to be accidental. The results of all available experiments in which the mixed arterial oxygen saturation was determined at least three times within the first hour of unilateral hypoxia are plotted in fig. 2. Conditions in these
Increased Pulmonary Vascular Resistance by Local Hypoxia 89 experiments were not uniform: the oxygen concentration of the low oxygen mixture varied from 0-22 to 541 per cent; the left lung was the test lung in seven experiments but the right lung in two; and, while the control lung was usually ventilated with pure oxygen, in one experiment air was used. In four of these nine curves exactly the expected type can be recognized. Two other experiments show a relatively high saturation, though not a strict maximum, at about 15 min. The fact that in the three remaining curves there was apparently no tendency towards an early maximum, was probably because the first sample had been drawn when there had already been some recovery from the fall normally observed within 2 or 3 min. A minimum was found between 25 and 65 min. in all nine experiments. In view of the difficulty of timing the samples so that they would coincide with the temporary maximum or minimum, the results agree surprisingly well with those obtained by the spirographic technique. We have only one experiment in which the mixed arterial oxygen tension was determined three times in the first hour [Dirken and Heemstra, 1948 a, p. 202]. Neither a maximum nor a minimum was found. In three out of seven other experiments of this type we did observe minima after a hypoxia of about I4 hrs. duration. Earlier minima in at least two of the remaining four experiments may well have escaped detection. Although the results just cited pointed somewhat to an absence or lessening of the diphasic response in the "oxygen tension" experiments, they cannot be regarded as conclusive because of too infrequent sampling. Furthermore, these experiments differ from the other ones in that the inspiratory oxygen concentration, instead of being below 5 per cent, varied between 13 and 17 per cent. Consequently a general hypoxaemia did not occur. DISCUSSION. The onset of the local increase in pulmonary vascular resistance is faster than might be expected, judging from the long period necessary to reach an approximately steady state. In this respect our results on rabbits agree with results of perfusion experiments and experiments on aniesthetized dogs. The diphasic character of the response cannot be explained as an artefact, due to the method of determination. If there is an error it is likely to be in the other direction, and the actual response in the first stage may have been much larger. If so, the diphasic character of the response will have been even more pronounced than the measurements show. Moreover, the results from the spirographic technique are confirmed by determination of mixed arterial oxygen saturation.
90 Heemstra The experiments with unilateral air breathing instead of 5 per cent oxygen show that the primary cause of the effect does not lie in the oxygen lung. This is in agreement with the results of Duke [1951], who did not observe in isolated perfused cat lungs a reaction to pure oxygen after air. Nevertheless, the vascular resistance in the oxygen lung will change passively as a result of the increased flow compensating for the decreased flow through the hypoxic lung. Rahn and Bahnson [1953] find that the hypoxic lung in dogs constricts less for a given alveolar PO2 when the control lung breathes air instead of 30 per cent oxygen. In our earlier work [Heemstra, 1948, Table 34] a similar observation was made. With the left lung breathing air and the right lung 6 per cent oxygen, during 8 hours the relative flow-ratio fell to only 74 per cent, which is above the range in our present experiments. The alveolar oxygen percentage in the control lung was reduced to 13-7 per cent, or almost 2 per cent below normal, due to its increased oxygen uptake. It was thought then that this change accounted for the slight increase of vascular resistance in the control lung, but it is perhaps not the only explanation of the phenomenon. The local effect in the nitrogen lung is very probably caused by the production of some substance which, in view of the slow recovery process, must be strongly fixed to the reacting tissue. Some may, however, be released into the circulation, and its effect on the control lung would then depend upon whether this lung breathes air or an oxygen-enriched mixture. The fact that the response is diphasic suggests that two processes are involved. As a working hypothesis it can be assumed that the basic reaction is a slow, nearly exponential rise of resistance which reaches its limit only after 8 hours. This main effect must be determined by a reduction in the calibre of the arteries or the capillaries and not in that of the venous side of the pulmonary circulation, since the reaction is accompanied by a definite paleness of the lung [Dirken and Heemstra, 1948 b]. Dye injection experiments point to the same conclusion. The "secondary" reaction determining the shape of the initial part of the curve may be a fast temporary vasoconstriction superimposed upon the slow one or, on the contrary, a later vasodilatation occurring in response to the initial primary vasoconstriction. Such an effect could be related to that described by Duke and Killick [1952], who found that CO depresses the pulmonary vascular resistance and that iodoacetate reverses the pressor response to anoxia. On the other hand, the oxygen lung may be concerned in the phenomenon in spite of the finding that oxygen itself has no appreciable effect on the lung vessels. More specifically one may think of a delayed vasoconstriction in this lung caused by the cumulative effect of a vasoconstrictor substance released into the circulation by the nitrogen lung. Finally, there is the additional possibility that an existing abnormal blood-flow ratio is modified by a non-specific general reaction such as a rise in pulmonary
Increased Pulmonary Vascular Resistance by Local Hypoxia 91 arterial pressure, or by a general change in vasomotor tone of the lung vessels or the release of some vaso-active substance elsewhere into the circulation. SUMMARY. An increase in pulmonary vascular resistance on unilateral breathing of 5 per cent 02 occurs in two distinct phases. The first maximum is reached in about fifteen minutes from the onset of hypoxia, after which the resistance falls until it reaches a minimum in the second half-hour. The second phase is represented by a gradual increase in resistance during the next eight hours, at the end of which the blood flow through the hypoxic lung is reduced to about 60 per cent of the original value. ACKNOWLEDGMENT. My thanks are due to Dr. Chr. L. Rfimke for help and advice in the statistical treatment of the results. REFERENCES. ATWELL, R. J., HIcKAM, J. B., PRYOR, W. W., and PAGE, E. B. (1951). Amer. J. Physiol. 166, 37. DIRKEN, M. N. J., and HEEMSTRA, H. (1948 a). Quart. J. exp. Phy8iol. 34, 193. DIRKEN, M. N. J., and HEEMSTRA, H. (1948 b). Quart. J. exp. Physiol. 34, 213. DUKE, H. (1951). Quart. J. exp. Physiol. 36, 75. DUKE, H., and KILLICK, E. M. (1952). J. Physiol. 117, 303. HEEMSTRA, H. (1948). Med. Dissertation. Groningen. KENDALT, M. G. (1948). Rank Correlation Methods, chapters 6 and 7. London: Charles Griffin & Co. Quoted from PH. VAN ELTEREN (1951). Report S59, Mathematical Centre, Amsterdam. NISELL, 0. I. (1950). Acta physiol. Scand. 21, Suppl. 73. PETERS, R. M., and Roos, A. (1952). Amer. J. Physiol. 171, 250. RAHN, H., and BAHNSON, H. T. (1953). J. Appi. Physiol. 6, 105.