J. Physiol. (1975), 250, pp. 463-473 With 4 text-figburem Printed in Great Britain

463

PULMONARY VASOMOTOR NERVE RESPONSES IN ISOLATED PERFUSED LUNGS OF MACACA MULATTA AND PAPIO SPECIES

BY THE LATE I. DE BURGH DALY, D. J. RAMSAY, AND B. A. WAALER* From the University Laboratory of Physiology, Parks Road, Oxford OX1 3PT

(Received 8 February 1974) SUMMARY

1. Lung lobes of Macaca mulatta and Papio species were isolated from the body and perfused by a pump delivering a constant volume inflow. The left atrial pressure was kept constant and therefore any recorded change in pulmonary arterial pressure reflected a change in pulmonary vascular resistance. 2. In five Macaca mulatta preparations stimulation of the upper thoracic sympathetic chain, the stellate ganglion, the middle cervical ganglion and the thoracic vagosympathetic nerve caused a small increase in calculated pulmonary vascular resistance usually followed by a larger decrease. Evidence is produced which suggests that the depressor response is mediated by adrenergic fl-receptors. In three preparations no change in pulmonary vascular resistance occurred. 3. In four Papio preparations stimulation of similar nerves invariably caused an increase in calculated pulmonary vascular resistance. In one animal no change in vascular resistance occurred. 4. A regression analysis of the results showed an inverse relationship between the magnitude of the pulmonary vascular response to nerve stimulation and the degree of excitement of the animals during capture, restraint and anaesthesia (P < 0.01). INTRODUCTION The main aim of the investigation was to determine whether functionally active sympathetic pulmonary vasomotor fibres, which in isolated perfused lungs of the dog cause a predominant increase in pulmonary vascular resistance (PVR) (Daly & Hebb, 1966; Daly, Ramsay & Waaler, 1970; Kadowitz & Hyman, 1973; Daly & Daly, 1973), are present in other * Present address: Institute of Physiology, University of Oslo, Norway. I7-2

I. DE BURGH DALY AND OTHERS mammalian species. For this purpose Macaca mulattt and Papio species were selected as the experimental animals. It has already been shown that isolated lungs of Macaca species can be successfully perfused for several hours (Daly, 1938; Hebb & Nimmo-Smith, 1948). 464

METHODS Experiments on Macaca mulaa and Papio 8pecw8 Anae8thesia. Eight Macaca mulatta (3-9-10.5 kg) and five Papio species (7010-0 kg) were either caught in a net or enticed into a small cage by a banana and, on entering, entrapped by a remotely controlled gate. The netted animals were given an i.v. injection of pentobarbitone (Nembutal, Abbott Laboratories Ltd) 22-29 mg/kg or an i.P. injection, 13-56 mg/kg. Alternatively, an I.M. injection of 3-5 mg/kg (phencyclidine hydrochloride Sernylan, Park Davis and Co.) was given. The caged animals were transferred to an anaesthetic box and anaesthesia was induced to the stage of muscular relaxation and loss of spinal reflexes with a nitrous oxide - oxygen (80v/20v) mixture passing through a modified Brodie (1902) chloroform vaporizer; i.v., I.P. or I.M. injections as mentioned above were then given. Two other kinds of anaesthesia were tried, without success. Oranges or bananas uniformly impregnated with pentobarbitone were rejected by the four animals tried. Attempts to shoot a pentobarbitone loaded syringe into the animals failed because, on presenting the syringe gun through the cage bars, they became extremely active and excited and presented a difficult target; this especially applied to the baboons. For this reason excitement was assessed in numerical terms, from a description of the excitement of each animal during each stage until the animal became unconscious. The score of 1 was given to those animals which were relatively quiet, and of 4 to those which were markedly active. Degrees of activity and excitement between these two extremes were given intermediate scores. Perfuwion of lung lobes. Directly the animals had been anaesthetized they were bled out and the procedures for ventilation and perfusion at a constant volume inflow were carried out as described by Daly, Michel, Ramsay & Waaler (1968). Both the pulmonary and bronchial circulations were perfused with either autologous blood alone, or a mixture of autologous blood with homologous blood or Dextraven (Benger Laboratories Ltd). There has been some confusion in the literature in that Ingram, Szidon, Skalak & Fishman (1968) have stated that a steady perfusion inflow has been used by Daly and his associates in earlier experiments. In fact a Dale-Schuster pump (1928) has been used for lung perfusion for the experiments in question (Daly, Duke, Hebb & Weatherall, 1948; Daly, Duke, Linzell & Weatherall 1952). This pump delivers a pulsatile outflow as is shown in Fig. 1 which is taken from an experiment on the left lung of a dog perfused at constant volume inflow using a Dale-Schuster pump (I. de B. Daly & M. de B. Daly, unpublished observations). Pulsations can be observed in the pulmonary arterial pressure and in the pulmonary arterial flow. As the perfusion set up in the experiments on Papio and Macaca mulata was identical to this, the flow in the experiments reported in this paper can be assumed to be

pulsatile. In Macaca mulatta and Papio species the left lung is comprised of three lobes although in the former species the separation between the apical and cardiac lobes may not be quite complete (Chase, 1942; Hayek, 1960). The right atrium was opened to allow free outflow of blood and thus to prevent any changes in right atrial pressure from causing passive effects on the Pp,, (Weil, Salisbury & State,

PULMONARY VASOMOTOR NERVE RESPONSES

465

1957). In some of the pulmonary nerve stimulation tests bronchial circulation perfusion was temporarily interrupted. Recording apparatus. The mean PPA was recorded by a damped Marey tambour located 20-25 cm above the level of the tip of the pulmonary arterial cannular opening. Alternatively the integrated mean PA derived electronically (ElemaSchonander, transducer EMT 490 B, 0-30 mmHg) was amplified by a R.C. conventional amplifier and recorded by a Sanborn electromagnetic pen-writer (Electronic and X-ray Applications Ltd) on 'Permapaper '. The pressure transducer was located 20-25 cm above the level of the pulmonary arterial cannula opening. With both methods a vertical 2 mm bore glass tube filled with saline or blood was connected in parallel with the recording apparatus so that the absolute mean PPA could be read on the attached scale. The oscillations within this tube were damped to enable the pressure to be read with ± 2*0 mm saline. The observed pressures were later converted to mmHg. The bronchial arterial pressure was recorded from the output of a 0-300 mmHg range Elema transducer. Tidal air changes were measured by the method of Konzett & Rossler (1940). Pulmonary vascular resistance (PVR). Because we have been unable to find data of the surface area of Papio species, the PVR has been calculated for both species in relation to their body weights, thus PVR

PPA(mmHg) - PLA(mmHg). Body Weight (kg)

PFlow (l./min) x where PpA and PLA = pulmonary arterial pressure and left atrial outflow pressure respectively, and x = ratio of total lung weight to the weight of the perfused lobe. The value of x was calculated on the assumption that the relative lung lobe weights of Macaca and Papio species were similar to those given by Rahn & Ross (1957) for the dog. This assumption might well mean that the calculated PVRs have a consistent error; they are, however, useful in comparing the PVRs in one experiment with another within each species group. Nerve stimulation. The topography of the left cardiopulmonary nerves in Macaca from our own observations is in agreement with that described by Zuckerman (1938). The middle cervical ganglion is represented by a plexus of several nodes receiving fibres from the stellate ganglion and the vagus. Caudal to this plexus, the issuing fibres join with other fibres from the stellate ganglion and vagus to form the thoracic vagosympathetic nerve. In Papio species, the topography of the left cardiopulmonary nerves closely followed that described by Mizeres (1955) for the dog. Stimulation of the left thoracic vagosympathetic nerve in both species was carried out with a pair of silver electrodes. At Attree (1950) square-wave stimulator was used with voltages varying from 5 to 100 V at 10 msec duration and 33-47 Hz, i.e. 5-100/10/33-47. These last two stimulation parameters were selected so that the pulmonary vascular responses obtained could be compared with those found in the dog in earlier experiments. In both species, because the nodes and nerve plexus proximal to the middle cervical ganglion were embedded in fat, in experiments involving stimulation of these parts of the nerve pathway, application of a solution of 1.0 % nicotine tartrate was used rather than electrical stimulation via the electrodes. Start of lung ventilation and perfusion. Five to ten minutes before the start of perfusion of the left apical and cardiac lobes or the whole left lung, 400 ml. of the pooled blood were circulated through the perfusion apparatus at approximately 100 ml./min. During the preparation for perfusion the collapsed lung lobes were kept moist by being covered in gauze soaked in physiological saline or by periodically

I. DE BURGH DALY AND OTHERS

466

spraying with warm saline. Initial ventilation pressures tend towards causing unequal expansion of the lungs and there is a danger of over-inflation of those portions already expanded if excessive pressure is used to expand the collapsed portions. Thus, damage to the capillaries and the small innervated blood vessels which supply and drain them (Fillenz, 1970) may take place. As a result of experience gained during the last 25 years or so, we have adopted a method of a gradual build-up of ventilation and perfusion which has not hitherto been described. The stroke volume of the Starling 'Ideal' respiration pump is initially set at a low value so that the lungs just start to move, and at the same time the stroke volume of the Dale-Schuster (1928) blood pump is gradually increased to give a recorded PpA of approximately 20 cm saline. Under these conditions the PpA gradually falls and as it does so the stroke volume of both the blood pump and the respiratory pump are increased until the PpA is brought back to 20 cm saline, care being taken not to exceed the normal expansion of the lungs at peak ventilation pressure. A progressive rise in PPA immediately demands a reduction in stroke volume of the blood pump. Because the blood flow through the lung lobes was kept constant, and the left atrial pressure was also constant, any change in mean PpA reflected a change in pulmonary vascular resistance across the lungs.

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L2

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Fig. 1. Pulsatile pressure and blood flow in an isolated left lung of a dog (body wt. 15-5 kg) perfused with blood at constant volume inflow through the pulmonary artery by means of a Dale- Schuster pump rotating at 102 c/mmn. Ventilation of lungs temporarily stopped in expiratory pressure 1.5 mmHg. Left atrial pressure held constant at 2H5 mmHg. Records from above downwards: QPA~pulmonary arterial blood flow (electromagnetic flowmeter type 372, Nycotron, Oslo, Norway). EPA, phasic pulmonary arterial pressure (Statham strain gauge transducer) and electrically integrated mean pressure (time constant 2 sec); and datum line. Time marker, 001 sec.

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467

RESULTS

Pulmonary vasomotor responses to nerve stimulation Macaca mulatta In three of the eight experiments performed, excitation ofthe pulmonary nerves had no effect on the mean PPA. The results obtained in the other five experiments are summarized in Table 1. Responses to nerve stimulation were only present during the first 60 min of perfusion. In all five animals, there was a small pressor effect, followed in four of them by a depressor effect. In three of the experiments, the depressor effect was more marked than the pressor effect. Such a response is illustrated in Fig. 2c. V15 ml.

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c b Fig. 2. Macacua mulatta, 7-5 kg. Non-atropinized isolated left lung. Pulsatile CVI perfusion through both the bronchial and pulmonary circulations at 102 and 117 c/min respectively. Blood temperature, 33.70 C. Left atrial blood flow, 225 ml./min; left atrial pressure, 2-0 mmHg; tidal air, 85 ml.; peak inflation pressure, 5*0 cm H20; expiratory pressure, 1-0 cm H20; respiratory pump cycles, 20/min; B.P. bronchial arterial pressure; PPA, mean pulmonary arterial pressure; VOV, ventilation overflow volume (Konzett & R6ssler, 1940), the lower portion of the trace has been removed. The first, second and third signal marks correspond to the first, second and third white dots respectively and indicate the interruption of bronchial circulation perfusion, the stimulus and the reintroduction of bronchial circulation perfusion respectively. a and b, the application of 0 05 m. 1 % nicotine tartrate solution to the stellate and middle cervical ganglion respectively. c, stimulation of the left thoracic vagosympathetic nerve at 10 V, 10 msec duration and 47 Hz (10/10/47).

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468 I. DE BURGH DALY AND OTHERS Fig. 3 demonstrates the effects of stimulation of the sympathetic nerves at various sites in their course to the lungs. As has been already mentioned, the thoracic vagosympathetic nerve was stimulated electrically and more proximal parts of the nervous pathway were stimulated with a 10% solution of nicotine tartrate. Each set of results from a single experimental animal are plotted on an unbroken base line. Although there are insufficient results to allow a full statistical analysis, it does appear that similar effects on pulmonary arterial pressure are obtained when the sympathetic nerves are stimulated at any part of their course to the lungs (Figs. 2 and 3). The Macaca

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response is usually a biphasic one, containing a depressor component. Also the pattern of response is similar in atropinized and non-atropinized preparations, with and without temporary interruption of bronchial circulation perfusion. These responses occurred therefore under conditions in which all known passive effects affecting the pulmonary circulation are excluded, and must be due to dilation of the pulmonary vessels themselves. Two experiments give some hint of the nature of the transmitter

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I. DE BURGH DALY AND OTHERS mediating the depressor response. Both were atropinized preparations and phenoxybenzamine had previously been added to the perfusion fluid. Thoracic vagosympathetic nerve stimulation caused a fall of PPA of 8-5 and 8 0 % (Fig. 3). After addition of pronethalol (Alderlin: A in Fig. 3), the response was abolished. The possibility arises that the vasodilator response is mediated by adrenergic fl-receptors. Papio 8pecie8 The pulmonary nerves were stimulated in five experiments, one of which failed to produce any pulmonary vasomotor responses. In the remaining four experiments, stimulation consistently caused a small rise in PPA (Fig. 3 and Table 1). In no experiment was there any evidence of a depressor response.

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As was the case in Macaca stimulation of any part of the course of the nerve elicited the response. In one experiment tried, phenoxybenzamine suppressed the rise in PPA elicited by nerve stimulation, which could be taken to support the hypothesis that the pressor effect depended on adrenergic a-receptors.

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Experiments which failed to respond to nerve stimulation on Macaca and Papio In searching for reasons for the absence of responses to nerve stimulation in four experiments we examined the following possibilities; the nature of the anaesthetic, the length of the ischaemic period to which the lung lobes were subjected before the start of perfusion, and the nature of the perfusate and its temperature during lung perfusion. No evidence was obtained that any one of these was a specific factor, although there was no certainty of this because of the small number of experiments involved. We did, however, find that when the results of the experiments on Macaca and Papio species were pooled there was an inverse relationship between the magnitude of the pulmonary vascular responses (constrictor or dilator) to nerve stimulation and the degree of excitement of the animals during capture, restraint and anaesthesia (Fig. 4). DISCUSSION

It is difficult to escape the conclusion that, when the sympathetic nerves to the lungs of Macaca are stimulated, there is usually a depressor component to the response. The response to nerve stimulation in Papio, however, is similar to that well known in the dog, i.e. an increase in PVR. The finding that the greater the degree of anxiety and excitement of the animals during capture and anaesthesia the less the responsiveness of the pulmonary vascular bed to nerve stimulation means that our results must be viewed with caution. It is true, that in Macaca subjected to a variety of forms of anaesthesia, a pulmonary vasodilator response to nerve stimulation in the isolated lung preparation predominated, but it is possible that only weak constrictor responses were recorded because the transmitter concerned in their production had been partially exhausted by the initial excitement of the animals and/or by anaesthesia. It might also be argued that the vaso-depressor response in Papio species was absent because the inhibitory transmitter had been exhausted under similar circumstances. The observations on Macaca lungs have a special significance in that, as far as we are aware, a response involving a reduction in PVR in response to pulmonary sympathetic nerve stimulation has not been reported as occurring in any other mammalian species. In the dog the most consistent response to pulmonary adrenergic fibre stimulation is a PVR increase (for literature before 1966, see Daly & Hebb, 1966; Daly et al. 1970; Pace, 1971; Kadowitz & Hyman, 1973; Daly & Daly, 1973). Rarely the response is a small decrease in PVR (Daly & Daly, 1958).

1. DE BURGH DALY AND OTHERS That the magnitude of the PVR response to sympathetic nerve stimulation in the dog is directly related to the frequency of stimulation up to 30 Hz has been reported by Kadowitz & Hyman (1973). This is in accord with our experience that stimulation frequencies of 50 Hz give the largest and most consistent pulmonary vasomotor responses (predominantly pressor) over the longest period of an experiment (Daly & Hebb, 1966, p. 401). It is also supported by an analysis of information from various sources, with the exception that with stimuli of 15-20 Hz Ingram et at. (1968) did not obtain any change in PVR. These investigators obtained evidence for a reduction in the compliance of the pulmonary arteries which was later (Ingram, Szidon & Fishman, 1970) supported by direct caliper measurements of the pulmonary artery diameter changes during nerve stimulation. Ingram et at. (1968) did not decentralize the stellate ganglion and the probability that changes in the cardiovascular system of the parent animal due to stimulation of its afferent nerves may have caused a reflex pulmonary vasodilation (Daly & Daly, 1961), thus masking the the pulmonary pressor response to efferent nerve stimulation, must be taken into account. The efferent nerve pathway for this reflex can be both ipsi- and contralateral (Daly & Hebb, 1954). Aarseth, Nicolaysen & Waaler (1971) using stimuli of 5-10 Hz also obtained results in agreement with those of Ingram et al. (1968) and only small or moderate increases in PVR. The finding that in isolated perfused lungs, sympathetic nerve stimulation may produce a depressor response in Macaca, and only pressor in Papio, seems clear. The physiological significance awaits clarification. 472

REFERENCES

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