Airway Hypocapnia Increases Microvascular Leakage in the Guinea Pig Trachea1- 3

ANN M. REYNOLDS, STEVEN P. ZADOW, RAFFAELE SCICCHITANO, and R. DOUGLAS MCEVOY

Introduction

We reported previously (1) that hypocapnia-induced bronchoconstriction in the guinea pig lung is mediated by tachykinins that are released after the activation of a bronchial axonal reflex. Tachykinins are a group of neuropeptides widely distributed in the central and peripheral nervous systems. In addition to smooth muscle constriction, tachykinins increase mucous gland secretion and cause postcapillary venule leakage and edema (2). Various inhaled substances, e.g., cigarette smoke and gastric acid, have been shown to produce vascular hyperpermeability in the airway by releasing tachykinins (3, 4). Airway hypocapnia is present in a number of physiologic and disease states (5, 6), and wetherefore wished to test whether airway hypocapnia could induce microvascular leakage by a similar mechanism. An in vivo method for isolating a tracheal segment was developed that allowed prolonged exposure of this segment to hypocapnic gas without causing adverse systemic effects. Vascular leakage was determined by the extravasation of intravenously injected Evans blue, an azo dye that binds avidly to circulating albumin (7). We have been able to show that airway hypocapnia increases microvascular leakage in the trachea. Tachykinins do not appear to be involved, but rather the change in vascular leakage appears to result from the production of prostaglandins and/or thromboxane. Methods Surgical Preparation Experiments were performed on Hartley strain guinea pigs of either sex weighing 540 ± 140 (SD) g. They were first anesthetized intraperitoneally with pentobarbital sodium (40 mg/kg) and placed on a heating blanket to maintain body temperature between 37and 390 C. Body temperature was monitored using a CIO series 80 temperature monitor connected to a rectal probe (Yellow Springs Instrument Co., Yellow Springs, OH). The trachea was then exposed just before it entered the thorax and cannulated with a 12-gauge plas80

SUMMARY We have previously shown that airway hypocapnia Induced bronchoconstrlctlon in the guinea pig lung by releasing tachykinins. To examine whether airway hypocapnia could also cause an Increase in airway microvascular leakage, a tracheal segment was Isolated in vivo In anesthetized guinea pigs and unldlrectlonally ventilated (200 ml/mln) for 1 h with fully conditioned air (0% CO.) or Isocapnlc gas (5% CO.). The lungs were ventilated through a distally placed tracheal cannula. Microvascular leakage was quantitated by the Injection of Evans blue (EB) and Its extraction from the tracheal segment. EB extravasation was Increased In tracheae exposed to 0% CO. (52.3 ± 2.0 ~g/g wet tissue) compared with tracheae exposed to 5% CO. (26.4 ± 2.9 ~g/g; P < 0.05) and to tracheae from spontaneously breathing guinea pigs (25.2 ± 2.3 ~g/g; P < 0.05). Groups of animals In which trachea were unldlrectlonally ventilated with 0% CO. were then pretreated With a range of drugs In an attempt to determine the mediators responsible for the microvascular leakage with 0% COa- Capsaicin and morphine pretreatment did not significantly alter 0% CO.-Induced EB extravasation, and phosphoramldon prevented rather than Increased extravasation, suggesting that tachyklnlns did not play a role. The hypocapnia-Induced Increase In microvascular leakage was, however,prevented by Indomethacin pretreatment and significantly attenuated by dazmegrel, a thromboxane synthetase Inhibitor. We conclude that airway hypocapnia causes microvascular leakage in the guinea pig trachea and that this effect is mediated by prostaglandins and/or thromboxane. AM REV RESPIR DIS 1992; 145:80-84

tic cannula (Dwellcath'"; Tuta Laboratories, Australia) through a longitudinal incision (approximately 3 to 5 mm) without ligation to minimize disruption to the tracheal microcirculation. This cannula was connected to a small animal ventilator (Model 665; Harvard Apparatus Co., South Natick, MA), and ventilation was commenced (30 to 33 strokesl min; tidal volume, 8 ml/kg; positive endexpiratory pressure, 2 em H 2 0 ) with air that was heated (370 C) and humidified (90070) as previously described (8). The animals were heparinized (150 U/kg) via a 20-gauge jugular vein catheter (Jelco'"; Jelco Corp., Raritan, NJ). This catheter was kept patent by filling its dead space with heparinized saline (150 U/ml) and used subsequently for Evans blue and intravenous drug administration (see below). A second 20-gauge fine cannula (Jelco") was passed through the anterior tracheal wall into the tracheal lumen just below the larynx and connected to a heated and humidified test gas source. This isolated tracheal segment (ITS) was ventilated unidirectionally with the test gas at a constant flow (200 ml/min) with gas escape at the distal cannula. Flow rate was measured continuously using a precision bore flowmeter (No. FP 118-080-5/81; Fischer & Porter, Workington, UK) connected in series with a compressed gas source. Reverse gas flow through the ITS was prevented by occluding the animal's nose and mouth with a rubber mask. All test gases used for unidirectional ventilation were heated

(390 C) and humidified (99%) prior to entering the trachea. Systemic arterial blood pressure was not routinely measured. Measurement of Plasma Extravasation Vascular leakage was determined by Evans blue (EB) extravasation using a modification of the method described by Saria and Lundberg (7). Immediately prior to unidirectional ventilation of the ITS, EB (50 mg/kg) was injected intravenously. After 1 h of unidirectional ventilation the animal was given a lethal intravenous injection of pentobarbital sodium. The chest was opened, and the heart was exposed. A 12-gauge plastic cannula was passed into the aorta and perfused with phosphate-buffered saline (pH, 7040) at a perfusion pressure of 100 mm Hg for 2.5 min to remove intravascular dye.The tracheal segment (approximately 2 em) was excised and (Received in original form October 11, 1990 and in revised form July 15, 1991) 1 From the Department of Thoracic Medicine, RoyalAdelaide Hospital, Adelaide, South Australia, Australia. a Supported by the Special Purposes Fund of the RoyalAdelaide Hospital and by the Asthma Foundation of South Australia. 3 Correspondence and requests for reprints should be addressed to A. M. Reynolds, Department of Thoracic Medicine, Royal Adelaide Hospital, North Tee., Adelaide S.A., Australia 5000.

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HYPOCAPNIA-INDUCED MICROVASCULAR LEAKAGE

dissected free of the esophagus and connective tissue. The middle third of this segment was then excised, blotted dry on filter paper, and weighed (Mettler balance Type H5). Evans blue was extracted in 2 ml formamide (Sigma Chemical Co., St Louis, MO) at 60° C for 24 h, and its concentration was determined by light absorbance at 620 nm (Varian DMS 80 spectrophotometer; Varian Associates, Palo Alto, CAl by comparison with the appropriate standard curve of EB in formamide (0.25 to 10 ng/ml). Tracheal EB content was expressed as micrograms per gram of wet tissue weight.

Experimental Groups Evans blue extravasation was examined in nine groups of animals. To determine the amount of dye extravasated in the trachea of animals on which no tracheal surgery was performed, spontaneously breathing animals (n = 6) were anesthetized intraperitoneally with a lower concentration of pentobarbital sodium (30 mg/kg) to prevent apnea, and they were then injected with EB and killed I h later. All other animals used in this study underwent the same surgical procedure (see above) and were subjected to I h of unidirectional tracheal ventilation. To test whether tracheal surgery itself induced vascular leakage, the ITS wasventilated for I h with isocapnic, normoxic gas (5OJo CO 2:21% O 2 ) in six animals. To test whether hypocapnia induced EB extravasation the ITS in another group (n = 6) was exposed to a hypocapnic normoxic gas (0% CO 2:21% O 2 ) . As a positive control the ITS (n = 3) was exposed to the irritant, capsaicin (20 nmol/kg; Fluka, Buchs, Switzerland) as an aerosol in isocapnic normoxic gas. This aerosol was generated by a Turret nebulizer powered at 8 L/min. To determine if tachykinins mediated any of the increase in microvascular leakage induced by hypocapnia (see RESULTS), a series of three experimental interventions wereused. (1) Tachykinin stores in peripheral nerve endings were depleted by repeated capsaicin injections as described previously (1). Briefly, capsaicin was dissolved in a vehicle containing ethanol-Tween 80 (3:1) and given subcutaneously over 5 consecutivedays in increasing doses (2 to 400 mg/kg). At least 7 days after the last injection the animals (n = 5) were prepared as described above, and the ITS was ventilated with 0% CO 2 gas. (2) The release of tachykinins from peripheral sensory nerve terminals was suppressed by morphine, which was infused intravenously 10 min prior to application of the hypocapnic stimulus (n = 6) at a dose of 10 mg/kg, which has been shown to significantly decrease neurogenic edema in the guinea pig airway (9). (3) The activity of any tachykinins that may have been released by the hypocapnic stimulus was enhanced by inhibition of neutral endopeptidase, the enzyme responsible for tachykinin degradation. In this experimental group (n = 5) phosphorarnidon (10-5 mol/kg; Sigma Chemical Co.) was infused over 3 min 5 min before hypocapnic ventilation of the ITS.

80 70

Fig. 1. Evans blue content expressedas micrograms per gram tracheal wet weight in trachea from spontaneously breathing control animals and animals exposed to 5% CO" 0% CO, and capsaicin aerosol. Values are mean ± SE. Asterisks indicate p < 0.05 compared with spontaneous breathing control animals.

60

50 40 30 20

10

In the final series of experiments we tested whether arachidonic acid metabolites could be implicated in hypocapnia-induced vascular leakage. The effect of cyclooxygenase inhibition on EB extravasation was studied by pretreating animals (n = 6) with indomethacin (40 mg/kg, dissolved in phosphate-buffered saline at pH 8.0) given intraperitoneally I h prior to surgery and hypocapnic challenge.Todetermine whether the microvascular leakage was mediated by thromboxane A" an additional six animals werepretreated with the thromboxane synthetase inhibitor, dazmegrel (UK-38485, a gift from Mr. A. C. Bostock, Pfizer, Sandwich, UK). Fresh solutions of dazmegrel were made up 30 min prior to use by dissolving 10 mg in 0.1 ml 1M NaOH and then adding 0.9 ml normal saline. Dazmegrel was then infused intravenously (10 mg/kg) 15 min prior to hypocapnic ventilation of the ITS.

Statistical Analysis Data are presented as the mean ± standard error. The Kruskal-Wallisanalysis of variance with multiple comparisons (10) was used to test whether significant differences existed between group mean values. A p value less than 0.05 was considered significant.

Results

The effect of airway hypocapnia on tra-

cheal EB extravasation is shown in figure 1.Hypocapnia (0070 CO 2 ) ventilation of the ITS significantly increased EB extravasation (52.3 ± 2.0 ug/g wet tissue, mean ± SE) compared with isocapnic (5% CO 2 ) ventilation (26.4 ± 2.9 ug/g; p < 0.05). Dye extravasation after isocapnic-ventilated trachea was not significantly different from that found in tracheae of spontaneously breathing animals (25.2 ± 2.3 ug/g). The addition of capsaicin aerosol to the isocapnic normoxie gas mixture significantly increased tracheal EB extravasation (61.9 ± 15.6 ug/g; p < 0.05) compared with 5% CO 2 ventilated trachea. Evans blue extravasation after ventilation with 0% CO 2 was not significantly different in the tracheae of capsaicinpretreated animals (59.2 ± 4.4 ~g/g) when compared with those from control animals (52.3 ± 2.0 ug/g) (figure 2). Morphine pretreatment before hypocapnic ventilation did not produce a significant decrease in dye extravasation either (44.1 ± 5.0 ug/g), However, phosphoramidon pretreatment significantly decreased dye extravasation (25.8 ± 3.8 ~g/g; p < 0.05) in trachea exposed to 0%

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Fig. 2. Tracheal Evans blue content after 0% CO, exposure in guinea pigs pretreated with capsaicin or after pretreatment with morphine, phosphoramidon, indomethacin, or dazmegrel. Values are mean ± SE. Asterisks indicate p < 0.05 compared with 0% CO,; dagger indicates p < 0.05 compared with indomethacin.

82

REYNOLDS, ZADOW, SCICCHITANO, AND MCEVOY

CO 2 to a level comparable with that in isocapnic-ventilated trachea. The effects of manipulating arachidonic acid metabolism on the increase in EB extravasation observed with 00/0 CO 2 tracheal ventilation are also shown in figure 2. Both indomethacin and dazmegrel pretreatment significantly attenuated vascular leakage induced by hypocapnic ventilation. With indomethacin treatment, EB extravasation (24.8 ± 3.2 ug/g) was not different from that in control (isocapnic)trachea (26.4 ± 2.9Ilg/g). With dazmegrel there was a significant decrease in extravasation (39.7 ± 2.1 Ilg/g) compared with control (0% CO 2 ) trachea, but the suppression was incomplete. Tracheal EB extravasation after indomethacin pretreatment was significantly lessthan that after dazmegrel (figure 2). Discussion

We have developed, in the guinea pig, a technique for isolating a section of trachea in vivo, similar to that described by other workers using larger species(11, 12). The advantage of this model is that gas composition within an isolated tracheal segment can be altered without affecting pulmonary gas exchange, and thus the effects of different gas concentrations on airway function can be examined in the absence of confounding systemic effects. Guinea pigs that underwent tracheal surgery followed by a l-h exposure of the ITS to isocapnic gas showed no difference in tracheal EB extravasation from that in anesthetizedspontaneously breathing animals. indicating that tracheal surgery in itself did not affect vascular leakage (at least in the middle one-third of the tracheal segment). The amount of tracheal EB extravasation in our animals exposed to isocapnic gas was comparable with EB extravasation data obtained by other workers (13, 14) in control guinea pigs. Our finding that capsaicin aerosol induced EB extravasation indicates that the ITS was responsive to an irritant stimulus and that the microcirculation after surgery was sufficiently intact to allow changes in protein extravasation to occur. Our main finding was that airway hypocapnia induced significant microvascular leakage in the guinea pig trachea. The use of air (0% CO 2 ) as the hypocapnic stimulus might appear unphysiologic. However, it should be noted that the trachea is a part of the airway that is already exposed to 0070 CO 2 during approximately half of the ventilato-

ry cycle(i.e.,inspiration). Although there was a doubling of EB extravasation with 0% CO 2 exposure, these changes are generally less than those reported after vagal nerve stimulation, toluene diisocyanate (TDI) inhalation, and substance P, capsaicin, or neurokinin A infusions (9, 13, 14, 15).

Critique of Method At anyone time the extent of EB extravasation in the trachea will be determined by the permeability of the tracheal microvasculature and by tracheal microvascular pressure (16). The latter will vary with changes in tracheal blood flow and vascular resistance. The changes we observed in EB extravasation in the present study could have been due to changes in vascular permeability, microvascularpressure, or both. Because we were unable to directly measure these parameters in the guinea pig trachea, wecannot be sure which mechanisms wereoperating. Measurement of systemic arterial pressure may have provided some information on possible hydrostatic pressure changes in the tracheal microcirculation. A possible criticism of our study is that systemic pressure was not measured. However, systemic arterial pressurewould be at best an indirect guide to tracheal microvascular pressure, and it is unlikely that such measurements would have allowed the mechanism of microvascular leakage to be determined. Although we do not have data from the present study on the hemodynamic effects of each of the pharmacologic agents used to investigate the mechanism of hypocapnia-induced microvascular leakage, wedo know that the drug that had the greatest inhibitory effect on EB extravasation (i.e., indomethacin) has been shown to have no effect on systemic arterial blood pressure in the guinea pig (17). Another possible weakness of the study is that we compared the effects of various pharmacologic agents on hypocapnia-induced microvascular leakage by reference to the results of a single control group of untreated animals. Because EB extravasation in untreated animals might vary from time to time (e.g., as a result of respiratory tract infection in the animal colony), it would perhaps have been more rigorous to have compared each pharmacologic intervention with its own hypocapnic control group. However, we conducted experiments on control animals (i.e., spontaneously breathing, isocapnia, and hypocapnia) contemporaneously with the pharmacologic studies.

Control experiments werespread over the entire experimental period, and control data showed the least between-animal variation (see figure 1). We believe that our experimental approach is unlikely to have biased our results or led to erroneous conclusions. A final possible criticism is that we did not test the effects of each of the pharmacologic agents on baseline (isocapnia) EB extravasation. For example, if one of these agents increased baseline (isocapnia) EB extravasation, it is conceivable that an inhibitory effect of that drug on hypocapnia-induced EB extravasation could have been masked. Wedid not look for such effects in the present study, but previous work has shown that capsaicin pretreatment, morphine infusion, phosphoramidon, indomethacin, and dazmegrel (UK 38485) have no effect on baseline plasma protein extravasation (9, 13, 18-20). We cannot, however, be entirely sure of the effect of dazmegrel diluent on baseline EB extravasation since the appropriate vehicle control experiment was not performed. Tachykinins Weinvestigated the role oftachykinin release in the pathogenesis of the hypocapnia-induced plasma leakage by a number of interventions. In one group, animals were systemicallypretreated with capsaicin in doses known to deplete sensory nerves to tachykinins (21), and in another, morphine was given to suppress tachykinin release from sensory nerve terminals (22). The absence of a significant protective effect on hypocapnia-induced microvascular leakage after these treatments provides strong circumstantial evidence that tachykinins do not mediate this vascular response. Phosphoramidon infusion, designed to enhance a response mediated by tachykinins (by prevention of their breakdown), actually prevented vascular leakage. Although phosphoramidon, a neutral endopeptidase inhibitor, has been shown to prolong the actions of tachykinins by inhibiting their degradation (23, 24), the action of this drug in vivo is likely to be complex because of its many substrates, which include bradykinin, vasoactive intestinal peptide (VIP), endorphins, and formyl-methionyl-leucyl-phenylalanine (25-27), and possibly calcitonin generelated peptide (CGRP) (28). One possible explanation that we have considered for the decrease in microvascular leakage with phosphoramidon is that it prevented EB extravasation by an indirect

83

HYPOCAPNIA·INDUCED MICROVASCULAR LEAKAGE

effect on arachidonic acid (AA) metabolism. For example, phosphoramidon reduces the breakdown of VIP (29) and CGRP (28). Given that hypocapnia is capable of releasing tachykinins in the guinea pig airway (1), it is conceivable that locally released VIP and CGRP remained in high concentrations after enkephalinase inhibition, thereby increasing intracellular cAMP levels (30, 31), which in turn reduces AA mobilization by inhibiting phospholipase A 2 (32). As outlined below, products of AA metabolism appear to play a key role in hypocapnia-induced vascular leakage. It is noteworthy that VIP is present in relatively large concentrations in guinea pig tracheae compared with tachykinins (33). Such an explanation is, however, quite speculative, and it could be equally argued that VIP and/or CGRP might be expected to increase microvascular leakage by producing vasodilation (34).

three being capable of inducing increased microvascular permeability. Therefore, it is possible that our results underestimate the role of 1XA2 in hypocapnia-induced vascular leakage, and the partial inhibition we observed after dazmegrelinfusion might have been the result of simultaneous increases in the levels of vasoactive prostanoids. Another possible explanation for an incomplete inhibition of hypocapnia-induced microvascular leakage by dazmegrel is that this drug may alter microvascular hemodynamics. Transient falls in systemicblood pressure have been reported with dazmegrel (39). If this were due to arteriolar vasodilation, transient increases in capillary and postcapillary venule leakage would be expected. It seems unlikely that the partial inhibition in microvascular leakage with dazmegrel was due to a partial pharmacologic effect on thromboxane synthetase. The intravenous dose used (10 mg/kg) was twice that known to inhibit in vivo Cyclooxygenase Products serum lXB2 (the stable metabolite of Indomethacin completely inhibited mi- TxA2 ) production by 750/0 in response to crovascular leakage after airway hypo- intravenously administered A-23187 in capnia, thereby strongly implicating cy- anesthetized guinea pigs (personal comclooxygenase products of AA as media- munication, Dr. M. J. Randall, Pfizer, tors. Indomethacin is a cyclooxygenase Central Research, Kent, UK). The way in which airway hypocapnia inhibitor that prevents the conversion of AA to thromboxane A 2 (TxA2 ) and the stimulates AA metabolism and increases prostaglandins PGI 2 , PGE 2 , PGD 2 and microvascular permeability was not adPGF2u • Thromboxane and the latter dressed in this study, and we can only three prostaglandins have been shown to speculate on possible mechanisms. Farincrease microvascular leakage (35,36). rukh and coworkers (40) showed that It is possible that indomethacin prevent- alkalosis (pH, 7.8), either by stimulating ed hypocapnia-induced extravasation by phospholipase A 2 (leading to a greater changing blood flow and driving pres- availability of AA) or by increasing the sures (factors known to effect plasma ex- activity of cyclooxygenase or thromboxudation) within the tracheal microcircu- ane synthetase, led to an increased prolation without there being an effect on duction of TxA2 in the rabbit pulmonary endothelial permeability. However, this circulation. The isolated enzyme system would appear an unlikely mechanism giv- of cyclooxygenase has been shown to en that a previous study (17) showed no have an optimal pH range of 7.7 to 8.2 change in mean systemicarterial pressure (41). Therefore, it is conceivable that airin guinea pigs pretreated with much high- way hypocapnia had its effect on the er doses of indomethacin (100 mg) than tracheal microcirculation by increasing were used in the present study. Further, mucosal pH, and thereby stimulating lowhen indomethacin was given in similar cal AA metabolism. Our previous work showed that airway doses to that used in this study, in the same species, no effect on baseline EB hypocapnia caused the releaseoftachykinins within the guinea pig airway, which extravasation occurred (19). The thromboxane synthetase inhibitor, resulted in marked bronchoconstriction dazmegrel, attenuated the vascular re- (1). Because tachykinins are known to sponse to hypocapnia, but not to the also cause microvascular leakage, we same degree as indomethacin. This indi- predicted that vascular leakage would cates that 1XA2 was at least in part re- also occur in the presence of airway sponsible for hypocapnia-induced micro- hypocapnia. The present results demonvascular leakage. Dazmegrel, while in- strated microvascular leakage in the trahibiting 1XA2 synthesis, simultaneously chea as predicted, but somewhat surprisaugments the production of PGI2 , POE 2 , ing was that AA metabolites rather than PGD 2 , and PGF2u (37, 38), the latter tachykinins appeared to be responsible.

This finding is analogous to that obtained by Ray and colleagues (42) who found that isocapnic hyperpnea-induced bronchoconstriction in the guinea pig was mediated by tachykinins, whereas the concomitant microvascular hyperpermeability appeared to be mediated by prostanoids (43, 44). Similarly, Thompson and coworkers (13)showed that in guinea pigs, TDI -induced bronchial hyperresponsiveness was mediated by tachykinins, whereas vascular leakage in the trachea following TDI exposure was not. The reason that tachykinins cause hypocapnia-induced bronchoconstriction, but seem not to be involved in hypocapnia-induced microvascular leakage in the trachea, may result from the differential distribution of neuropeptides within guinea pig airways. Tachykinins are present in relatively small concentrations in the trachea compared with the major and segmental bronchi (15, 45). Therefore, it is likely that any stimulus that results in tachykinin releasewill have its effect in the main bronchi and distal airways. In our previous study (1), we reported that airway narrowing after hypocapnia was restricted to distal airways, consistent with the distribution of tachykinins within guinea pig airways. In summary, airway hypocapnia induced significant microvascular leakage in the guinea pig trachea. The effect was inhibited by indomethacin and a thromboxane synthetase inhibitor, suggesting that hypocapnia-induced vascular leakage is mediated by prostaglandins and/or thromboxane. These results, combined with our previous findings (1), suggest that a fall in CO 2 concentration in the airways can lead to the release of a variety of chemical mediators that may have deleterious effects on airway function. References 1. Reynolds AM, McEvoy RD. 'Iachykinins mediate hypocapnia-induced bronchoconstriction in guinea pigs. J Appl Physiol 1989; 67:2454-60. 2. Martling C-R. Sensory nerves containing tachykinins and CGRP in the lower airways. Functional implications for bronchoconstriction, vasodilation and protein extravasation. Acta Physiol Scand Suppl 1987; 130:563:5-57. 3. Lundberg JM, Saria A. Capsaicin-induced desensitization of airway mucosa to cigarette smoke, mechanical and chemical irritants. Nature 1983; 302:251-3. 4. Martling C-R, Lundberg JM. Capsaicin sensitive afferents contribute to acute airway edema following tracheal instillation of hydrochloric acid or gastric juice in the rat. Anesthesiology 1988; 68:350-5. 5. McFadden ER, Lyons HA. Arterial blood gas tensions in asthma. N Engl J Med 1968; 278: 1027-32.

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