RESPIRATORY MODULATION BY VAGAL AFFERENTS IN PIGS

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1 Quarterly Journal of Experimental Physiology (1981) 66, Printed in Great Britain RESPIRATORY MODULATION BY VAGAL AFFERENTS IN PIGS Institute of Veterinary Physiology and Biochemistry, via Celoria 10, Milan and Institute of Human Physiology, via Mangiagalli 32, Milan (RECEIVED FOR PUBLICATION 6 JUNE 1980) SUMMARY We have studied the role of pulmonary vagal afferents and baroreceptors of the aortic arch on control of breathing in anaesthetized pigs. As in other species (dogs, cats and rabbits) the depth and rate of breathing was found to be controlled by a volume-related vagal feed-back loop from pulmonary stretch receptors. Rapid shallow breathing following histamine aerosol and phenyldiguanide I.V. (which are likely to stimulate pulmonary vagal iritant and J receptors) occurred through a decrease in the activation threshold of the inspiratory cut-off mechanism without altering the matching of inspiratory to expiratory time. These results are also similar to those found in cats and rabbits. Histamine caused an increase in pulmonary resistance and a decrease in compliance: these effects were partially vagally mediated and partially due to a direct stimulation of smooth muscles by the drug. It seems, from relating the respiratory to the vascular systemic effect of histamine administration, that pigs have a greater respiratory response than cats or rabbits. A stepwise decrease in blood pressure through haemorrhage under iso-pc02 and iso-po2 conditions was found to affect the matching of the expiratory to the inspiratory time without affecting the activation threshold of the inspiratory cut-off mechanism. INTRODUCTION In recent years the pig has become increasingly important in cardiovascular physiology and experimental biology in general (Bustad & McClellan, 1965; Bustad, McClellan & Burns, 1966; Pond & Houpt, 1978) as it was shown to be closer to humans than to most other mammals (Luginbuhl, 1966; Martin, 1964; Roberts & Strauss, 1965; Englehardt, 1966; Rowsell, Mustard, Packham & Dodds, 1966; Maaske, Booth & Nielsen, 1966). Little is known, however, concerning its respiratory physiology, in particular the respiratory reflexes and control of breathing which have been extensively studied recently in other experimental animals like cats, rabbits and dogs. In the present investigation we have evaluated in pigs the role of vagal afferents of various origin on control of breathing pattern. We considered pulmonary vagal afferents from the three known types of receptors; stretch, irritant and J receptors (Paintal, 1973). Stretch receptors were shown to be involved in the respiratory phase-switching mechanisms in anaesthetized cats (Clark & Euler, 1972; Euler, 1977); rabbits (Miserocchi, Trippenbach, Mazzarelli, Jaspar & Hazucha, 1978) and dogs (D'Angelo, Miserocchi & Agostoni, 1976). Stimulation of irritant and J receptors was found to result in a strong tachypneic effect (Karczewski & Widdicombe, 1969, in rabbits; Winning & Widdicombe, 1976; Miserocchi et al. 1978; Miserocchi, Mazzarelli, Quinn & Mozes, 1979, in cats) with an increase in pulmonary resistance and compliance (Karczewski & Widdicombe, 1969). Moreover we studied the ventilatory response to a decrease in blood pressure which was shown to be related to a decrease in baroreceptor afferents from the aortic arch and to hypoxic stimulation of glomus cells (Miserocchi & Quinn, 1980).

2 264 METHODS Experiments were done on nineteen pigs of either sex weighing kg, anaesthetized with pentobarbitone sodium (30-40 mg/kg) injected into the cranial cava vein through the jugular sulcus. The animals were premedicated with 005 ml./kg of % propyonylpromazine given intramuscularly, which quietened them sufficiently to enable the anaesthetic injection to be made with minimal restraint by hand. Supplementary doses of pentobarbitone sodium were given, when necessary, through a catheter inserted into the femoral vein. The animals were tied out supine and body temperature was measured with a rectal probe and maintained at the physiologic level (37-38 C) by an electric blanket. Catheters were inserted into the right femoral artery and into the jugular vein, the latter being advanced into the right atrium. Systemic arterial blood pressure (B.P.) was recorded by connecting the femoral catheter to a fluid filled capacitance manometer (Bell & Howell 4-422). Mean pressure was obtained from pulsatile signal by electrical average. After tracheotomy, a tracheal cannula was inserted just below the cricoid cartilage; tracheal pressure was measured with a Bell & Howell pressure transducer connected to a side arm of the tracheal cannula. Air flow was obtained using a Fleish pneumotachograph head No. 2; electrical integration provided tidal volume. Records of systemic arterial blood pressure, tidal volume and tracheal pressure were made after suitable amplification on a Grass poligaph (model 7 B). The respiratory responses to histamine (H) and phenyldiguanide (PDG) were studied in eight pigs. In other species these drugs were shown to stimulate pulmonary vagal irritant and J receptors. Histamine was delivered as aerosol by passing a flow of compressed air through a commercial generator containing a 02-05% (usually 02%) solution of histamine in saline. Phenyldiguanide was injected into the right atrium ( ,g in 0-6 ml. of saline solution). To avoid tachyphylaxis, the drugs were administered at intervals of not less than min. In these animals lung resistance and compliance were also measured by the 'subtractor' method of Mead & Whittenberger(1953) modified by Green & Widdicombe(1966). To this end transpulmonary pressure (tracheal minus intrapleural pressure) was measured between an air-filled polyethylene catheter tied into a lower right intercostal space, and the side arm of the tracheal cannula, using a Bell and Howell differential capacitance manometer. The effects of these drugs were also studied after vagosympathectomy performed bilaterally in the mid-cervical region. In this region the vagus nerve joins the sympathetic trunk and they are held in a close association by a common connective tissue sheath down to the level of the seventh cervical vertebra; before cutting the nerve a few drops of carbocaine were applied to the vagus nerve. Experimental procedure involving vagosympathectomy was not started until min after cutting the nerve. The role of vagal afferents from the aortic arch was studied by decreasing blood pressure in five pigs through progressive bleeding through the left femoral artery. The respiration was monitored with animals in control conditions and at different values of blood pressure reached during bleeding; usually 5-10min were necessary to reach a steady state respiratory pattern at each value of blood pressure. In these animalspa, c0pa 02 and ph were measured by blood gas analyser (Instrumentation Laboratory 413). Since a drop in blood pressure was accompanied by hyperventilation causing hypocapnia,co2 was delivered to the animal so as to maintainpa co, at a value similar to control condition. + The total volume of shed blood was 25 2(S.E.M.) ml./kg body weight. In four animals bilateral vagotomy was performed in the neck after the bleeding had been stopped (at a mean blood pressure of - 45 mmhg). In six more pigs acute vagotomy was performed at normal blood pressure. Analysis of the data. The spontaneous pattern of breathing was analysed in (TI and TE, Fig. 1). terms of tidal volume (VT) and inspiratory and expiratory times In addition we also evaluated the timing of breaths following occlusion of the airways at the end-expiratory level (Fig. 1). As suggested by Grunstein, Younes & Milic-Emili (1973), the comparison between the spontaneous pattern of breathing and the one following occlusion allows an estimate of the reflex role of the phasic lung volume related vagal informations. Using a differential block technique (Miserocchi et al. 1978) most of these informations in cats and rabbits were shown to be from pulmonary stretch receptors. The occluded inspiratory time was obtained from the tracheal pressure record from the onset of the inspiratory effort until peak negative pressure developed in the airways (Fig. 1, TO) and the occluded expiratory time from peak value to the onset of the next breath (Fig. 1, Five to ten respiratory cycles were analysed both for unoccluded and occluded breaths. TO)).

3 VAGAL AFFERENTS IN PIGS 265 Intact B.P. 100 r - Vagotomy (m m Hg) 0,,,.,,..~TP c (cmh20) 1 s 0 VT 10[ L (T TE (U * EM M M N A M ~ M M Fig. 1. Experimental records of blood pressure (B.P., upper tracing), of tracheal pressure (Tp, middle tracing) and tidal volume (VT, lower tracing), before and after vagotomy during control conditions and following histamine and phenyldiguanide (PDG) stimulation. Inspiratory (TI) and expiratory (TE) times are shown on tidal volume tracing for unoccluded breaths. Inspiratory and expiratory times for occluded breaths (T, and TO respectively) are shown on tracheal pressure record. RESULTS Fig. 1 shows the experimental records for blood pressure, tracheal pressure and tidal volume obtained for control conditions and after histamine (H) and phenyldiguanide (PDG) stimulation before and after vagotomy. H aerosol caused rapid shallow breathing within 5 min and this pattern remained unaltered as long as the aerosol was maintained. PDG caused a short-lasting ( 5 sec) apnea followed by rapid - shallow breathing which lasted about 12 sec. As this time was very short several injections were usually necessary to collect data both for unrestricted and occluded breaths. Fig. 2 shows the effect of H (triangle) and PDG (square) on various respiratory parameters before and after vagotomy. With intact vagi, H and PDG shortened the inspiratory (A) and expiratory (B) duration of occluded (half-filled symbols) and unoccluded breaths (filled symbols); moreover they decreased tidal volume (C). The only significant effect of drug stimulation after vagotomy was an increase in tidal volume caused by PDG. Vagotomy caused an increase in tidal volume and in inspiratory time. The post-vagotomy inspiratory time was essentially equal to the prevagotomy duration of inspiration during occluded breaths. Conversely the post-vagotomy duration of expiration was significantly shorter (08+0±38 (S.E.) sec) than the prevagotomy occluded expiratory time. Before vagotomy H and PDG decreased the end-expiratory value of oesophageal pressure (data from 4 animals) by ±2 (S.E.) cm H20 and 02 +0±2 (S.E.) cm H20 respectively. Neither

4 266 2 A 3B4 ~12 E X 16 Ẹ ~2-2 1 ntact 0 3u nac u A 170 ntc0u A Vagi Vagi Vagi Vagi Vagi Vagi intact cut intact cut intact cut Fig. 2. Mean values from all the pigs for Tn(A), TE (B) and VT(C) for prevagotomy unoccluded (open symbols), occluded (half-filled symbols) and post-vagotomy (closed symbols) breaths during control (0) and the following histamine (A) and phenyldiguanide stimulation (l). Bars refer to SE. of the average change as compared to control A B U 20E20.* - lo 100 Vagi Vagi Vagi Vagi intact cut intact cut Fig. 3. Effect of histamine and phenyldiguanide on total lung resistance (A) and compliance (B) before and after vagotomy; symbols as in Fig. 2. Points represent the mean values for all the animals studied; bars refer to S.E. of the average change as compared to control. change was significant, but, owing to the measured compliance of pig's lungs (47 5 ml./cm H20), such changes would have decreased the end-expiratory volume by 16 ml. Fig. 3 A - shows that H caused a marked increase in airways resistance both before and after vagotomy although the prevagotomy effect was significantly greater than the post-vagotomy one. Conversely PDG did not affect airways resistance. Fig. 3 B shows that H and PDG decreased the compliance markedly before vagotomy; this effect being greater for H. After vagotomy H still caused a decrease in compliance but the effect was significantly less than the prevagotomy one, Vagotomy caused a significant decrease in resistance ( (S.E.) cm H20/l.sec) and an increase in compliance ( (S.E.) ml./cm H20). In control conditions B.P. was mmhg and heart rate (H.R.) was cycles/min. H and PDG decreased B.P. by (S.E.) and (S.E.) mmhg and heart rate by (S.E.) and (S.E.) cycles/min respectively. We should recall that H was shown to cause a byphasic effect on blood pressure as an initial short lasting (5 sec) decrease was followed by a subsequent increase to a steady value lower than the control one (Lorenz, Barth, Kusche, Reimann, Schmal, Matejka, Mathias, Hutzel & Werle, 1971). We refer to this steady phase.

5 VAGAL AFFERENTS IN PIGS A I A I A B.P. (mmhg) B B.P. (mmhg) Fig. 4. A, inspiratory (TI) and, B, expiratory (TE) durations of occluded breaths plotted as a function of blood pressure from five animals. In A: TO = B.P., P > In B: TO = B.P., r = 0-66, P < Closed circles are mean values for four animals after vagotomy. Vagosympathectomy did not significantly affect B.P. and H.R. After vagosympathectomy H and PDG did not significantly change B.P. and H.R. The fact that the post-vagotomy expiratory time was shorter than the prevagotomy occluded expiratory time indicated that vagal afferent activity inhibitory to respiratory frequency was tonically present at the end-expiratory volume. These data were confirmed by performing acute vagotomy in six more pigs at an average blood pressure of (S.E.) mmhg; this caused a significant reduction in TO( (S.E.) sec) with no change in TO. Data from Fig. 4 show that on progressively decreasing blood pressure there was a significant shortening of the occluded expiratory time with essentially no change in the occluded inspiratory time. Vagotomy performed in four animals at the end of the bleeding procedure (average blood pressure (S.E.) mmhg) decreased, though not significantly, the expiratory time (closed circle) by sec. The overall shortening of TO on decreasing blood pressure from to - 50 mmhg was 0-7 sec, i.e. similar to that obtained on performing acute vagotomy at normal blood pressure. We were not fully successful in adjusting the CO2 delivery to the animals so as to maintain isocapnic conditions. Indeed for the five pigs subject to haemorrhage we found that at a B.P. of (S.E.) mmhg, Pac0,, Pa 02 and ph were torr; torr; whilc at a B.P. of mmhg the corresponding values were torr, torr and respectively. Despite these difference in P.,co, no significant

6 I Inspiratory time (sec) Inspiratory time (sec) A-f I C D 3-2 I 3-6 u 2-8 W * *f 2 x ,- t u 3-2 E * *!; 2-4 x 2 1 I I 0 0* Inspiratory time (sec) Inspiratory time (sec) Fig. 5. A, VT vs. TI relationship obtained by joining the co-ordinates of unloaded (open symbols) to those of loaded breaths (half-filled symbols) before and after vagotomy (closed symbols). Control conditions (0); exposure to histamine aerosol (A); i.v. injection of phenyldiguanide (O). Points refer to mean values from eight animals. B, VT vs. T1 relationship obtained as in A at control blood pressure (0 0, B.P. = mmhg), with decreased blood pressure following bleeding (0----0,.P. = 533 +I 1 mmhg), and after subsequent vagotomy (. 0, B.P. = mmhg). Prevagotomy points refer to mean values from five animals; postvagotomy points refer to mean values from four animals. C, expiratory vs. inspiratory time relationships obtained by joining the co-ordinates of unloaded (open symbols) to those of loaded breaths (half-filled symbols) for control condition (circles), for histamine (triangles) and phenyldiguanide (squares). Closed symbols are mean values after vagotomy. D, Expiratory vs. inspiratory time relationships at a B.P. 111 mmhg (i) and at B.P. = 53 mmhg (ii). Closed circle refers to post-vagotomy point. All bars refer to S.E. of mean. relationship was found betwen the values of TO and the corresponding values of kco2. Vagotomy did not significantly affect blood gases and ph. Fig. 5A and B shows the tidal volume vs. inspiratory time relationship obtained under different conditions. Such a relationship has been proposed as a model regulating the depth and rate of breathing as it would describe the interaction between the threshold sensitivity curve of the inspiratory cut-off mechanism and the afferents from pulmonary stretch receptors (Euler, 1977). These relationships were approximated by joining with a straight line the co-ordinates of the unoccluded to those of the occluded breaths (Miserocchi et al.

7 VAGAL AFFERENTS IN PIGS ). The relationships have a negative slope meaning that progressively less afferent input from pulmonary stretch receptors is needed to cut short inspiratory activity as time progresses. Fig. 5 A shows that before vagotomy H and PDG displaced the tidal volume vs. inspiratory time relationships leftward. Vagotomy (closed symbols) abolished the negative slope of the relationship according to the suppression of the phasic vagal input from pulmonary stretch receptors: H and PDG did not significantly alter post-vagotomy data. Fig. S B shows that decreasing blood pressure did not alter the tidal volume vs. inspiratory time relationship. Fig. S C and D shows the expiratory vs. inspiratory time relationships obtained under various conditions. These relationships were obtained by joining with a straight line, under each condition, the co-ordinates of the unoccluded to those of the occluded breaths (Miserocchi et al. 1978). They provide an estimate of how the phasic vagal afferents from pulmonary stretch receptors affect the respiratory phase-switching mechanisms. Fig. SC shows that H and PDG had no significant effect on the slope and intercept of this relationship; post-vagotomy points (closed symbols) are also shown and are clearly displaced downward as compared to the prevagotomy relationship. Fig. 5D shows that decreasing blood pressure from I ±424 (S.E.) to (S.E.) mmhg significantly decreased the position and the slope ofthe relationship so that the post-vagotomy point (closed symbol) lay close to the relationship referring to a low blood-pressure value. The dashed line refers to hypoxic response data obtained in a previous research and it will be dealt with in the discussion. DISCUSSION In analogy with what happens in other species (dogs, cats and rabbits), in pigs the phasic afferents from pulmonary vagal receptors are involved in the mechanism cutting short inspiratory activity (as shown by the negative slope of the VT vs. T1 relationship (Fig. S A) and relating expiratory to inspiratory time (as shown by the TE vs. T, relationship (Fig. 5C). As in other previously studied species, i.e. cats and rabbits (Winning & Widdicombe, 1976; Miserocchi et al. 1978, 1979) H and PDG caused rapid shallow breathing in pigs and this reflex was essentially abolished by vagotomy. The increase in VT caused by PDG after vagotomy stems from an excitatory effect on the respiratory output (indexed by the inspiratory flow) of either central origin or, in analogy with what is found in cats (Dawes, Mott & Widdicombe, 1951; Dawes & Comroe, 1954), of stimulation of peripheral chemoreceptors. Since before vagotomy this effect was not present this indicates that vagal afferents stimulated by PDG have an opposite (inhibitory) effect on respiratory output in line with what was found by Miserocchi et al. (1979). As in cats and rabbits, drug stimulation resulted in a marked lowering of the volume threshold for inspiratory cut-off (leftward displacement of the VT vs. T, relationship, Fig. 5 A) without greatly altering the matching of expiratory to inspiratory time (Fig. S C). These effects have been attributed to the stimulation of pulmonary vagal irritant and J receptors within the lung as, from single fibre recording, these drugs were shown to greatly increase the activity of these receptors (Paintal, 1973). Although such recordings were not done in pigs it seems likely that there is a common role played by these receptors among species as far as the reflex respiratory pattern is considered. Differences exist, however, when the dose-response ratio is considered. Indeed the doses giving the maximum respiratory response in pigs were ten times smaller (both for H and PDG) than those used in cats and rabbits (Miserocchi et al. 1979).

8 270 Table 1. Comparison of effects of histamine on lung compliance and conductance in cats (from Colebatch et al. 1966), dogs (from De Kock et al. 1966), rabbits (from Karczewski et al. 1969) and pigs Decrease in compliance (0) Decrease in conductance (0) Species Dose Before vag. After vag. Before vag. After vag. Cat 6-48,ug/kg I.V. Dog 3-24,ug/kg I.V. Rabbit 100 fig/kg I.V. Pig aerosol 01-05gg% Histamine caused a marked increase in respiratory resistance with a decrease in compliance. At least 50%O of this effect was vagally mediated while the other 50%o was due to a direct stimulating action of the drug on the smooth muscles of the airways. Comparative values for the reflex changes in respiratory conductance and compliance in response to histamine stimulation are given in Table 1 for cats, dogs and rabbits. Reflex changes appear to be much greater in pigs as compared to the other species. We shall note, however, that in pigs histamine was delivered as aerosol while in the other species it was given intravenously. We calculated that if all the histamine delivered to the animal had been absorbed this would have represented a dose of- 100,tg/kg. This value, compared with those reported for the other species, would confirm a greater histamine sensitivity in pigs in terms of respiratory reflexes. This is at variance with a reported low vascular systemic reactivity (Lorenz et al. 1971). The difference between the respiratory and the vascular reactivity to histamine is also confirmed by relating in different animals the respiratory timing effect (indexed by A TTO.t = ATI + A TE) to the vascular effect (indexed by A B.P.). The ATTO0t/AB.P. ratio is about 500 times greater in pigs than in cats and rabbits. One should add here that in pigs the histamine content in whole blood is the second highest in all the mammals studied (Lorenz & Werle, 1969; Lorenz et al. 1971) and it is also relatively high in all the tissues including the lung (Lorentz et al. 1971). PDG caused no change in resistance but only a decrease in compliance. This effect, which seems to be vagally mediated as it disappears after vagotomy and is similar to the one described by Colebatch, Olsen & Nadel (1966) and De Kock, Nadel, Zwi, Colebatch & Olsen (1966) for histamine stimulation in vagotomized cats and dogs respectively. It was ascribed to a specific action of the drug on the most peripheral airways. This effect, due to PDG, is peculiar to pigs as compared to cats and rabbits. A decrease in blood pressure obtained through stepwise haemorrhage did not affect the volume threshold curve (Fig. SB; no significant changes in the co-ordinates of unrestricted and loaded breaths) but greatly affected the matching of expiratory to inspiratory time (Fig. SD). In particular the decrease in blood pressure from -110 to 50 mmhg reduced TF more than T, as shown by a reduction in the slope of the relationship between these two variables. This frequency inhibitory vagal-dependent effect was greatly reduced (as shown by results from vagotomy (Fig. 4)) when B.P. was - 50 mmhg. A decrease in afferents from aortic

9 VAGAL AFFERENTS IN PIGS baroreceptors may explain this finding, as afferents from these receptors were shown to reduce respiratory frequency (Heymans & Neil, 1958; Miserocchi & Quinn, 1980). On the other hand a possible decrease in functional residual capacity occurring during haemorrhage may have caused a decrease of the tonic firing of pulmonary stretch receptors which was shown to shorten the timing of breaths in the absence of phasic lung volume-related informations in other species (Bartoli, Bystrzycka, Guz, Jain, Noble & Trenchard, 1973; Sant'Ambrogio, Camporesi, Sellick & Mortola, 1972; Phillipson, 1974; Miserocchi et al. 1978). Haemorrhage implies hypoxic stimulation of peripheral chemoreceptors owing to a reduced blood flow (Daly, Lambertsen & Schweitzer, 1954; Neil, 1951; Purves, 1970; McCloskey & Torrance, 1971) as early postulated by Comroe (1939). However, recently Aguggini, Clement & Davies (1979) found that hypoxic stimulation in pigs did not affect the expiratory vs. inspiratory time relationship (see Fig. SD) so that the observed vagal dependent phenomenon cannot be explained by this mechanism. The effects ofhaemorrhage on control of breathing have been recently studied in cats and at variance with pigs it was found that a decrease in blood pressure markedly decreased the volume threshold for inhibition of inspiratory activity without greatly affecting the TE vs. TI relationship (Miserocchi & Quinn, 1980). Thus, although the general effect of decreasing blood pressure is an excitatory one on breathing frequency this occurs through different mechanisms acting on the respiratory phase-switching controlling systems in cats and pigs. 271 REFERENCES AGUGGINI, G., CLEMENT, M. G. & DAVIES, A. (1979). Unusual response of anaesthetised pigs to asphyxia. Research in Veterinary Science 26, BARTOLI, A., BYSTRZYCKA, E., Guz, A., JAIN, S. K., NOBLE, M. I. M. & TRENCHARD, D. (1973). Studies on the pulmonary vagal control of central respiratory rhythm in the absence of breathing movements. Journal of Physiology 230, BUSTAD, L. K. & MCCLELLAN, R. 0. (1965). Use of pigs in biomedical research. Nature 208, BUSTAD, L. K., MCCLELLAN, R. 0. & BURNS, M. P. (1966). Swine in Biomedical Research. Richland, Washington: Pacific Northwest Laboratory. CLARK, F. J. & EULER, C. VON. (1972). On the regulation of depth and rate of breathing. Journal of Physiology 222, COLEBATCH, H. J. H., OLSEN, C. R. & NADEL, J. A. (1966). Effect of histamine, serotonin and acetylcholine on the peripheral airways. Journal of Applied Physiology 21, (1), COMROE, J. H. J. (1939). The location and function of the chemoreceptors of the aorta. American Journal of Physiology, 27, DALY, M. DE B., LAMBERTSEN, C. J. & SCHWEITZER, A. (1954). Observations on the volume of blood flow and oxygen utilization of the carotid body in the cat. Journal of Physiology 125, D'ANGELO, E., MISEROCCHI, G. & AGOSTONI, E. (1976). Effect of ribcage or abdomen compression at iso-lung volume on breathing pattern. Respiration Physiology 28, DAWES, G. S. & COMROE, J. H., JR. (1954). Chemoreflexes from the heart and lungs. Physiological Reviews 34, DAWES, G. S., MOTT, J. C. & WIDDICOMBE, J. G. (I1951). Respiratory and cardiovascular reflexes from the heart and lungs. Journal of Physiology 115, DE KOCK, M. A., NADEL, J. A., Zwi, S., COLEBATCH, H. J. H. & OLSEN, C. R. (1966). New method for perfusing bronchial arteries: histamine bronchoconstriction and apnea. Journal of Applied Physiology 21, (1), ENGLEHARDT, W. V. (1966). Swine cardiovascular physiology. In Swine in Biochemical Research, ed. BUSTAD, L. K., MCCLELLAN, R. 0. & BURNS, M. P., p Richland, Washington; Pacific Northwest Laboratory.

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