European Journal of Pharmacology, 215 (1992) 51-56 ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
51
EJP 52433
Effect of maturation on histamine-induced airflow obstruction and airway microvascular leakage in guinea pig airways Hirokazu Arakawa, Kenichi Tokuyama, Tatsuya Yokoyama, Hiroyuki Mochizuki, Akihiro Morikawa, T a k a y o s h i K u r o u m e a n d P e t e r J. B a r n e s ~' Department (~f Pediatrics, Gunrna Unit,ersity School of Medicine, Maebashi, Gunma 371, Japan and ~ Department of" Thoracic Medicine. National Heart and Lung Institute, London SW3, U.K. Received 14 November 1991, revised MS received 6 February 1991, accepted 18 February 1992
To study the effect of maturation on histamine-induced airflow obstruction and airway microvascular leakage, we measured concomitant changes in lung resistance (R L) and in extravasation of Evans Blue dye in the airways of anesthetized immature (aged 14 + 2 days) and adult guinea pigs (aged 60 _+ 12 days). R L was measured for 6 min after iv. histamine (0, 5, 15, 30 and 50 /zg/kg). For comparison, responses after 1 p.g/kg substance P were also examined. After measurement of RE, microvascular leakage in trachea, main bronchi, and proximal and distal intrapulmonary airways was also examined in the same animal. Immature animals required a larger dose of histamine than adults to achieve a similar degree of maximal bronchoconstriction after histamine. In contrast, equal doses of histamine (15 and 30 /zg/kg) induced a significantly greater extravasation of dye in immature airways in both proximal and distal intrapulmonary airways, although not in trachea or main bronchi. Substance P did not cause any age-related differences in dye extravasation at any airway level. These results suggest that i.v. histamine specifically causes a greater degree of airway microvascular leakage in peripheral airways but induces less smooth muscle contraction in the airways of immature guinea pigs than in the airways of adult animals. Aging; Airway obstruction; Asthma; Oedema; Histamine; Plasma exudation; Substance P; Vascular permeability
1. Introduction
Airway microvascular leakage is a feature of airway inflammation and may have a pathophysiological role in asthma (Barnes, 1989; Persson, 1988). Airway microvascular leakage may result in airway edema and contribute to mucus plug formation, and thus may contribute to airflow obstruction or bronchial hyperresponsiveness (Barnes, 1988; Hogg et al., 1987; Persson, 1988). Exuded plasma components such as bradykinin or anaphylatoxin might contribute to local inflammation by activating inflammatory cells or neural pathways in the airways. Histamine, a well characterized spasmogen for airway smooth muscle, is also potent in inducing airway microvascular leakage when given either intravenously (Evans et al., 1989; Saria et al., 1983) or by aerosol (Tokuyama et al., 1991a, b, c) to experimental animals, including rats and guinea pigs. Recently, age-related
Correspondence to: K. Tokuyama, Department of Pediatrics, Gunma University School of Medicine, Maebashi 371, Japan. Tel. 81.272.31 7221 ext. 3271, fax 81.272.34 2039.
differences in histamine-induced airway responses have been demonstrated in guinea pig, swine and sheep airways by comparing the changes in lung resistance or by direct m e a s u r e m e n t of airway caliber. These age-related differences include the maximum degree of bronchoconstriction (Clerici et al., 1989a; Murphy et al., 1991; Sauder et al., 1986), major reactive sites in the airways (Murphy et al., 1991), and the involvement of cholinergic neural pathways in the contractile response (Clerici et al., 1989a). It is possible that histamine-induced airway microvascular leakage may also change with age, since microvascular leakage may be a component of airflow obstruction when histamine is given by aerosol (Tokuyama et al., 1991a, b, c). The purpose of this study was to examine the agerelated differences in histamine-induced airflow obstruction and airway microvascular leakage in the same animal. We measured concomitant changes in lung resistance (R E) and airway microvascular leakage after i.v. histamine in immature and adult guinea pig airways. For comparison, responses after substance P, a potent mediator of airway microvascular leakage (Lundberg et al., 1984; L6tvall et al., 1990), were also examined.
52 2. Materials and methods
Experiments were performed on 70 pathogen-free male D u n k i n - H a r t l e y guinea pigs divided into two age groups (Clerici et al., 1989a, b): immature (200 _+ 18 g; 14 _+ 2 days) and adult (481 _+ 71 g; 60 _+ 12 days) animals. Animals were anesthetized with an initial dose of urethane (6-8 m l / k g ; 25% w / v in 0.9% saline) injected intraperitoneally (i.p.). Additional urethane was given as required to maintain anesthesia. A tracheal cannula (10 m m length and 2.7 mm inner diameter in adults and 8 mm length and 2.0 m m inner diameter in immature animals) was inserted into the lumen of the cervical trachea through a tracheostomy and secured with a suture. A polyethylene catheter was inserted into the left carotid artery to monitor blood pressure and heart rate with a pressure transducer. The right external jugular vein was cannulated for the administration of histamine, substance P or their vehicle (0.9% NaC1) and Evans Blue dye.
2.1. Measurement of airway function Guinea pigs were placed in a supine position with the intratracheal cannula connected to a constant volume mechanical ventilator (Model SN-480-7; Shinano Co., Tokyo, Japan). A tidal volume of 10 ml per kg and a frequency of 60 breaths per minute were used. Transpulmonary pressure was measured with a pressure transducer (Model TP-603T; 50 c m H 2 0 ; Nihon Koden Co., Tokyo, Japan), with one side attached to a catheter inserted into the right pleural cavity and the other side attached to a catheter connected to a side port of the intratracheal cannula. The ventilatory circuit had a total volume of 18 ml. Airflow was measured with a pneumotachograph (Model TU-241T; Nihon Koden Co., Tokyo, Japan) connected to a transducer (Model TP-602T; 5 cm H 2 0 ; Nihon Koden Co., Tokyo, Japan). All signals were recorded on a 4-channel recorder (WT-645 GC; Nihon Koden Co., Tokyo, Japan). Lung resistance (R L) was calculated as described previously (Von Neergaard and Wirz, 1927).
2.2. Protocol I m m a t u r e or adult guinea pigs were divided into six groups each (total 12 groups) in order to compare the effects of serially diluted histamine (5, 15, 30 and 50 /xg/kg), 1 # g / k g substance P or their vehicle (0.9% NaC1) on airway microvascular leakage and increases in lung resistance for 6 min after administration. Ten minutes after connection to the ventilator, Evans Blue dye (20 m g / k g ) was administered i.v. One minute later, histamine was administered i.v. R L, transpulmonary pressure and mean blood pressure were recorded every
minute. Five minutes after drug administration, the animals were hyperinflated with twice the tidal volume by manually blocking the outflow of the ventilator, and 1 min later disconnected from the ventilator. We defined R L at 6 min as 'recovery R L' (L6Wall et al., 1990; Tokuyama et al., 1991a, b, c), because hyperinflation would be expected to diminish airway smooth muscle contraction and abolish airflow obstruction induced by atelectasis.
2.3. Determination of plasma extrat,asation After disconnection from the ventilator, the thoracic cavity was opened, and a cannula was inserted into the aorta through the left ventricle. Animals were perfused with 0.9% NaCI at a pressure of 100 mm Hg, to remove intravascular dye. The lungs were removed, and the lung parenchyma carefully scraped off. The trachea, main bronchi and intrapulmonary airways were separated from each other, and the intrapulmonary airways were divided into two equal portions lengthwise, arbitrarily named proximal and distal. All tissues were weighed wet. Evans Blue dye was extracted in 2 ml of formamide at 40°C for 24 h and measured in a spect r o p h o t o m e t e r (UV-150-02; Shimazu Co., Kyoto, Japan) at 620 nm. The extracted Evans Blue dye was quantified by interpolation on a standard curve of dye concentrations in the range of 0.5-10 ~zg/ml, and expressed as ng d y e / m g tissue. Evans Blue dye extravasation has been shown previously to correlate with the extravasation of radiolabelled albumin in guinea pig airways (Rogers et al., 1989).
2. 4. Drugs and chemicals The following drugs and chemicals were used: urethane and histamine (Kanto Chemical Co., Tokyo, Japan); Evans Blue dye (Aldrich Chemical Co., Milwaukee, USA); 0.9% NaCI (Fuyo Chemical Co., Osaka, Japan); substance P (Peninsula, USA).
2.5. Data analysis Data are reported as means_+ S.E.M. A M a n n Whitney U-test was used to test for significant differences between individual groups. A P value less than 0.05 was considered significant.
3. Results
3.1. Comparison of lung resistance Baseline R L w a s significantly higher in immature animals than in adults in all of the groups studied (fig. 1). In both immature and adult animals, histamine
53 TRACHEA
BASELINE
~oo~~z~
~
6O 40
~
E
t
g
"i"E u v
~
MAXIMAL RESPONSE
0L
E
Control
5
15
30 Histamine [#g,/kg]
50
MAIN BRONCHI
.~
8O 6O
--
~. I°°I ~
0
1
Substance P (#g/kgl
Fig. 1. Changes in lung resistance after i.v. histamine in immature and adult guinea pigs. Baseline (upper panel) and maximum increase in Rt. (lower panel) after control (0.9% NaCI), 5, 15, 30 and 50 /zg/kg histamine and 1 / z g / k g substance P in immature ( I ; n = 5, 5, 6, 5, 5 and 4, respectively) and adult guinea pigs ( [] ; n = 6, 7, 10, 6, 6 and 5, respectively) are shown. * P < 0.05 and ** P < 0.01 when comparing immature and adult animals receiving equal doses of histamine or substance P. + P < 0.05 and ++ P < 0.01 when compared with control animals of the same age.
40
~ m ~> to
PROXIMAL-IPA
80 60 40
20
~
o
DISTAL-IPA
m
~
0
caused a dose-dependent increase in R L, with the maximal response occurring within seconds after administration. The minimum dose of histamine to induce a significant increase in R e was 1 5 / z g / k g in both age groups. Substance P, 1 /zg/kg, did not cause a significant increase in R e compared to vehicle in either age group. The maximal response to 15, 30 and 50 / z g / k g histamine was greater in adults than in immature animals, with a significant difference at 5 0 / z g / k g histamine (fig. 1). Histamine, 50 /zg/kg, caused a marked fall in blood pressure and death in adults. In immature animals, it induced a higher value of the recovery R e (0.72 + 0.03) after histamine compared to the baseline R L (0.60 _+ 0.03), although this was not significant (0.05 < P < 0.1). In animals receiving 5, 15 or 30 / z g / k g histamine or its vehicle, recovery R g returned to baseline levels in both immature and adult animals.
3.2. Comparison of extraL:asation of Et,ans Blue dye At all airway levels, no significant difference was found in the extravasation of Evans Blue dye after vehicle (0.9% NaCI) between the immature and adult animals (fig. 2). Histamine caused a dose-dependent increase in the amount of dye exuded in both groups of animals. The minimum concentrations to induce significant extravasation of dye were 5 / z g / k g at trachea, 15 / z g / k g at main bronchi and proximal and distal intrapulmonary airways in immature animals, and 15 # g / k g at all airway levels in adults. We could not compare the degree of extravasated dye after 50 / x g / k g histamine, because this dose of histamine caused a marked fall in
i]
t
5
Control
15
30
Histamine [ ug/kg)
1
Substance P [,ug/kg)
Fig. 2. Airway microvascular leakage after different dose of histamine. Extravasation of Evans Blue dye at different airway levels after control (0.9% NaCI), 5, 15 and 30 /zg/kg histamine and 1 /zg/kg substance P in immature ( I ) and adult guinea pigs (12) are shown. Extravasation of dye in animals receiving 5 0 / x g / k g histamine could not be compared because many animals died, especially adult animals. * P < 0.05 and ** P < 0.01 when comparing immature and adult animals receiving equal doses of histamine or substance P. • P < 0.05 and ** P < 0.01 when compared with control animals of the same age. Numbers in columns show numbers of experimental animals in each group. IPA: intrapulmonary airways.
blood pressure and death trachea or main bronchi, differences in the amount mature and adult animals
in most adult animals. In there were no significant of dye exuded between imfor matched doses of his-
TABLE 1 Percent change in mean systemic blood pressure after i.v. administration of histamine in immature and adult guinea pigs. Values are means + S.E.M.; a p < 0.05 compared to control animals of the same age. No significant difference was found in the decrease in systemic blood pressure between immature and adult animals after the same doses of histamine. ( - ) : impossible to evaluate. Animals
Control (0.9% NaCI) Histamine 5 (/zg/kg) 15 30 50
Immature
Adult
1.8-+ 1.0 - 1.3 + 4.3 - 15.5 _+1.9 " - 17.2_+3.0 a - 17.6_+3.1 "
1.4_+2.0 -2.6_+2.6 - 11.4_+4.7 a -11.5_+5.1 a
(-)
54 tamine. In contrast, in proximal and distal intrapulmonary airways, there was significantly more dye exuded in immature animals after 15 or 30 /.tg/kg histamine than in adult animals. Substance P caused a significantly greater increase in dye extravasation at all airway levels than vehicle (0.9% NaCI). No significant differences in the amount of dye was observed at any airway levels between immature and adult animals after substance P (fig. 2).
3.3. Changes in blood pressure I.v. histamine caused a dose-dependent fall in systemic blood pressure (table 1). At 5, 15 and 30 p.g/kg, there were no significant differences in the decrease in blood pressure between immature and adult animals receiving corresponding doses of histamine. Histamine, 50/.tg/kg, induced a marked fall in blood pressure and death in adult animals. Substance P caused a marked fall in systemic blood pressure in both immature and adult animals ( - 5 9 . 5 + 3.0 and - 3 7 . 0 _+ 9.7% compared to the baseline levels, respectively), although there was no age-related difference.
4. Discussion
We have shown that immature guinea pigs required a larger dose of histamine than adults to achieve a similar degree of bronchoconstriction. In contrast, the degree of extravasation of Evans Blue dye after histamine was significantly greater in immature airways, especially in the periphery, while this was not the case after substance P. In both immature and adult animals, histamine caused a dose-dependent increase in RL, with maximal responses occurring within seconds after drug administration. Although the minimum dose to cause a significant increase in R L was not different between the two groups (15 /.tg/kg), the maximal response in R L was greater in adults, with a significant difference at 50 / z g / k g histamine. As observed previously (Clerici et al., 1989a, b), baseline R L was significantly higher in immature animals, presenting smaller airway diameters in resistance airways than in adults. A decrease in initial airway diameters has been shown to cause a greater increase in airflow resistance to stimuli, because resistance is inversely proportional to the fourth power of the airway radius (Barnes et al., 1988a). Therefore, our results suggest that smooth muscle in immature airways is less reactive to histamine, confirming previous observations in guinea pig (Clerici et al., 1989a), swine (Murphy et al., 1991) or sheep airways (Sauder et al., 1986). The lack of a vagal component in immature animals (Clerici et al., 1989a) and changes in the density or coupling of histamine H l receptor or in the
distribution of the two different histamine receptor subtypes (Ahmed et al., 1980) have been suggested to explain this age-related difference in response to histamine. Another possible explanation for this age-related difference is a decrease in the quantity of airway smooth muscle in immature airways, as has been described in human airways (Matsuba and Thurlbeck, 1972). In the present study, a concentration of substance P of 1 ~ g / k g was insufficient to cause a significant increase in R L in both age groups although it increased the extravasation of Evans Blue dye, confirming previous observations that substance P is much more potent in inducing airway microvascular leakage than in causing airway smooth muscle contraction (Rogers et al., 1988). In both age groups studied, histamine caused a dose-dependent increase in airway microvascular leakage at all airway levels. We could not compare the degree of extravasation after 50 /~g/kg histamine, because at this dose histamine caused a marked fall in blood pressure and death in adult animals. In contrast to trachea or main bronchi, where no significant difference was observed, a significantly greater dye extravasation was seen in most peripheral airways of immature animals after 15 or 30 g g / k g histamine. These results indicate that histamine induces a greater degree of microvascular leakage in immature airways, especially in peripheral airways. We do not know the mechanism of this airway level-dependent difference in microvascular leakage. Age-related changes in blood flow in peripheral airways might be a reason for this difference, because blood flow could be a major determinant in the degree of microvascular leakage (Persson, 1987). In contrast to histamine, substance P induced a greater extravasation of dye in adults in trachea and main bronchi, but caused no difference between adults and immatures in intrapulmonary airways. This result suggest that factors other than changes in blood flow with maturation might underlie an age-related difference in histamine-induced airway microvascular leakage, especially in peripheral airways. Changes in the density of histamine receptors or in the distribution of two different histamine receptor subtypes located in the microvenules might also contribute to this age-related difference. It has been shown that part of the plasma extravasation induced by histamine is mediated via sensory nerves (Lundberg and Saria, 1983). Therefore, the development of sensory nerves might be related to this difference. Murphy et al. (1991) have shown that, in young swine, i.v. histamine causes a significantly greater narrowing of airway diameter at peripheral airways, but not at central airways, than in adult animals. Airway edema resulting from airway microvascular leakage could be one of the contributory factors for this airway level-dependent difference in airway narrowing.
55
We have previously shown that histamine, when given by aerosol, causes a rapid increase in R L, peaking within seconds, followed by a partial recovery within minutes, with a prolonged elevation in R e which persists after hyperinflation of the lung (Tokuyama et al., 1991a, b, c). Although the initial increase is likely to reflect airway smooth muscle contraction in view of its rapid time course, we speculated that the more sustained phase was related, at least in part, to airway wall edema resulting from airway microvascular leakage, because the amount of dye exuded was significantly correlated with R e after hyperinflation (recovery R L) (L6tvall et al., 1990; Tokuyama et al., 1991a, b, c). In the present study, i.v. histamine caused a rapid increase in R t, which peaked within seconds, similar to the result obtained with aerosol administration. However, recovery R e in most animals returned to baseline levels. The difference in recovery R L between our previous observation (Tokuyama et al., 1991a, b, c) and present results may be due to the different routes of administration. Various pathogenic effects of histamine have been shown including anaphylaxis (Barnes et al., 1988). As shown in animals receiving 50 ~ g / k g histamine, i.v. administration might cause more severe cardiovascular responses than by aerosol administration, which would limit a large increase in R e or airway microvascular leakage. Indeed, the peak increase in R L was lower than in our previous study in which histamine was administered by aerosol (Tokuyama et al., 1991a, b, c). Histamine, as well as substance P, induced a significant decrease in systemic blood pressure in both immature and adult animals. Because changes in systemic blood flow may also affect the degree of microvascular leakage (Persson, 1987), changes in systemic blood pressure might be a cause of the age-related difference in the amount of dye exuded after histamine. However, this is unlikely, because no significant difference in changes in systemic blood pressure was observed between immature and adult animals receiving the same doses of histamine or substance P. We have shown that, in immature guinea pigs, the degree of airway microvascular leakage caused by histamine is greater, especially in the peripheral airways, while airway smooth muscle is less reactive to histamine than in adult guinea pigs. We do not know whether similar changes during maturation operate in human airways. Histamine has been shown to be a mediator of airway inflammation, including allergic inflammation (Barnes et al., 1988b). Therefore, if this were the case in man, airway edema or mucus plugs resulting from airway microvascular leakage might be greater in airways of young children than in adults during an asthmatic attack caused by several stimuli such as allergens, in spite of a lower responsiveness of the airway smooth muscle.
Acknowledgement We would like to thank Dr. Tetsuhiro Kubota, Kissei Pharmaceutical Company, Matsumoto, Japan, for his support of our experiments.
References Ahmed, T., P. Lyre, A.J. Januszkiewicz and A. Wanner, 1980, Role of H I- and H2-receptors in airway reactions to histamine in conscious sheep, J. Appl. Physiol. 49, 826. Barnes, P.J., 1989, A new approach to the treatment of asthma, N. Engt. J. Med. 321, 1517. Barnes, P.J., I.W. Rodger and N.C. Thomson, 1988a, Pathogenesis of asthma, in: Asthma: Basic Mechanism and Clinical Management, eds. P.J. Barnes. 1.W. Rodger and N.C. T h o m s o n (Academic Press, London) p. 415. Barnes, P.J., K.F. Chung and C.P. Page, 1988b, Inflammatory mediators and asthma, Pharmacol. Rev. 4(1, 49. Clerici, C., A. Hart. C. Gaultier and F. Roudot, 1989a, Cholinergic component of histamine-induced bronchoconstriction in newborn guinea pigs, J. Appl. Physiol. 66. 2145. Clerici, C., I. Macquin-Mavier and A.A. Hart, 1989b, Nonadrenergic bronchodilatation in adult and young guinea pigs, J. Appl. Physiol. 67, 1764. Evans, T.W., D.F. Rogers, B. Aursudkij, K.F. C h u n g and P.J. Barnes, 1989, Regional and time-dependent effects of inflammatory mediators on airway microvascular permeability in the guinea pig, Clin. Sci. 76, 479. Hogg, J.C., P.D. Pare and R. Moreno, 1987, The effect of submucosal edema in airways resistance, Am. Rev. Respir. Dis. 135, $54. L6tvall, J.O., R.J. Lemen, K.-P. Hui, P.J. Barnes and K.F. Chung, 1990, Airflow obstruction after substance P aerosol: Contribution of airway and pulmonary edema, J. Appl. Physiol. 69, 1473. Lundberg, J.M. and A. Saria, 1983, Capsaicin-induced desensitization of the airway mucosa to cigarette smoke, mechanical and chemical irritants, Nature (London) 302, 251. Lundberg, J.M., E. Brodin, X. Hua and A. Saria, 1984, Vascular permeability changes and smooth muscle contraction in relation to capsaicin-sensitive substance P afferents in the guinea-pig, Acta Physiol. Scand. 120, 217. Matsuba, K. and W.M. Thurlbeck, 1972, A morphometric study of bronchial and bronchiolar walls in children, Am. Rev. Respir. Dis. 105, 9(/8. Murphy, T.M., L, Roy, I.J. Phillips, R,W. Mitchell, E.A. Kerry, N.M. Mufioz and A.R. Left, 1991, Effect of maturation on topographic distribution of bronchoeonstrictor responses in large diameter airways of young swine, Am. Rev. Respir. Dis. 143, 126. Persson, C.G.A., 1987, Leakage of macrornolecules from the tracheobronchial circulation, Am. Rev. Respir. Dis. 135, $71. Persson, C.G.A., 1988, Plasma exudation and asthma, Lung 166, 1. Rogers, D.F., M.G. Belvisi, B. Aursudkij, T.W. Evans and P.J. Barnes, 1988, Effects and interactions of sensory neuropeptides on airway microvascular leakage in guinea-pigs, Br. J. Pharmacol. 95, 1109. Rogers, D.F., P. Boschetto and P.J. Barnes, 1989, Plasma exudation: correlation between Evans Blue dye and radiolabelled albumin in guinea-pig airways in vivo, J. Pharmacol. Meth. 21,309, Saria, A., J.M. Lundberg, G. Skofitsch and F. Lembeck, 1983, Vascular protein leakage in various tissues induced by substance P., capsaicin, bradykinin, serotonin, histamine and by antigen challenge, Naunyn-Schmiedeb. Arch. Pharmacol. 324, 212.
56 Sauder, R.A., K.J. McNicol and A.A. Stecenko, 1986, Effect of age on lung mechanics and airway reactivity in lambs, J. Appl. Physiol. 61, 2074. Tokuyama, K., J.O. L6tvall, C.-G. L6fdahl, P.J. Barnes and K.F. Chung, 1991a, Inhaled formoterol inhibits histamine-induced airflow obstruction and airway microvascular leakage, Eur. J. Pharmacol. 193, 35. Tokuyama, K., J.O. L6tvall, P.J. Barnes and K.F. Chung, 1991b, Mechanism of airway narrowing caused by inhaled platelet-
activating factor: Role of airway microvascular leakage, Am. Rev. Respir. Dis. 143, 1345. Tokuyama, K., J.O. L6tvall, A. Morikawa, P.J. Barnes and K.F. Chung, 1991c, Role of thromboxane A 2 in airway microvascular leakage induced by inhaled platelet-activating factor, J. Appl. Physiol. 71, 1729. Von Neergaard, K. and K. Wirz, 1927, Die Messung der Stromungswiderstande in den Atemwegen des Menschen, insbesondere bei Asthma und Emphysem, Z. Klin. Med. 105, 51.
51
EJP 52433
Effect of maturation on histamine-induced airflow obstruction and airway microvascular leakage in guinea pig airways Hirokazu Arakawa, Kenichi Tokuyama, Tatsuya Yokoyama, Hiroyuki Mochizuki, Akihiro Morikawa, T a k a y o s h i K u r o u m e a n d P e t e r J. B a r n e s ~' Department (~f Pediatrics, Gunrna Unit,ersity School of Medicine, Maebashi, Gunma 371, Japan and ~ Department of" Thoracic Medicine. National Heart and Lung Institute, London SW3, U.K. Received 14 November 1991, revised MS received 6 February 1991, accepted 18 February 1992
To study the effect of maturation on histamine-induced airflow obstruction and airway microvascular leakage, we measured concomitant changes in lung resistance (R L) and in extravasation of Evans Blue dye in the airways of anesthetized immature (aged 14 + 2 days) and adult guinea pigs (aged 60 _+ 12 days). R L was measured for 6 min after iv. histamine (0, 5, 15, 30 and 50 /zg/kg). For comparison, responses after 1 p.g/kg substance P were also examined. After measurement of RE, microvascular leakage in trachea, main bronchi, and proximal and distal intrapulmonary airways was also examined in the same animal. Immature animals required a larger dose of histamine than adults to achieve a similar degree of maximal bronchoconstriction after histamine. In contrast, equal doses of histamine (15 and 30 /zg/kg) induced a significantly greater extravasation of dye in immature airways in both proximal and distal intrapulmonary airways, although not in trachea or main bronchi. Substance P did not cause any age-related differences in dye extravasation at any airway level. These results suggest that i.v. histamine specifically causes a greater degree of airway microvascular leakage in peripheral airways but induces less smooth muscle contraction in the airways of immature guinea pigs than in the airways of adult animals. Aging; Airway obstruction; Asthma; Oedema; Histamine; Plasma exudation; Substance P; Vascular permeability
1. Introduction
Airway microvascular leakage is a feature of airway inflammation and may have a pathophysiological role in asthma (Barnes, 1989; Persson, 1988). Airway microvascular leakage may result in airway edema and contribute to mucus plug formation, and thus may contribute to airflow obstruction or bronchial hyperresponsiveness (Barnes, 1988; Hogg et al., 1987; Persson, 1988). Exuded plasma components such as bradykinin or anaphylatoxin might contribute to local inflammation by activating inflammatory cells or neural pathways in the airways. Histamine, a well characterized spasmogen for airway smooth muscle, is also potent in inducing airway microvascular leakage when given either intravenously (Evans et al., 1989; Saria et al., 1983) or by aerosol (Tokuyama et al., 1991a, b, c) to experimental animals, including rats and guinea pigs. Recently, age-related
Correspondence to: K. Tokuyama, Department of Pediatrics, Gunma University School of Medicine, Maebashi 371, Japan. Tel. 81.272.31 7221 ext. 3271, fax 81.272.34 2039.
differences in histamine-induced airway responses have been demonstrated in guinea pig, swine and sheep airways by comparing the changes in lung resistance or by direct m e a s u r e m e n t of airway caliber. These age-related differences include the maximum degree of bronchoconstriction (Clerici et al., 1989a; Murphy et al., 1991; Sauder et al., 1986), major reactive sites in the airways (Murphy et al., 1991), and the involvement of cholinergic neural pathways in the contractile response (Clerici et al., 1989a). It is possible that histamine-induced airway microvascular leakage may also change with age, since microvascular leakage may be a component of airflow obstruction when histamine is given by aerosol (Tokuyama et al., 1991a, b, c). The purpose of this study was to examine the agerelated differences in histamine-induced airflow obstruction and airway microvascular leakage in the same animal. We measured concomitant changes in lung resistance (R E) and airway microvascular leakage after i.v. histamine in immature and adult guinea pig airways. For comparison, responses after substance P, a potent mediator of airway microvascular leakage (Lundberg et al., 1984; L6tvall et al., 1990), were also examined.
52 2. Materials and methods
Experiments were performed on 70 pathogen-free male D u n k i n - H a r t l e y guinea pigs divided into two age groups (Clerici et al., 1989a, b): immature (200 _+ 18 g; 14 _+ 2 days) and adult (481 _+ 71 g; 60 _+ 12 days) animals. Animals were anesthetized with an initial dose of urethane (6-8 m l / k g ; 25% w / v in 0.9% saline) injected intraperitoneally (i.p.). Additional urethane was given as required to maintain anesthesia. A tracheal cannula (10 m m length and 2.7 mm inner diameter in adults and 8 mm length and 2.0 m m inner diameter in immature animals) was inserted into the lumen of the cervical trachea through a tracheostomy and secured with a suture. A polyethylene catheter was inserted into the left carotid artery to monitor blood pressure and heart rate with a pressure transducer. The right external jugular vein was cannulated for the administration of histamine, substance P or their vehicle (0.9% NaC1) and Evans Blue dye.
2.1. Measurement of airway function Guinea pigs were placed in a supine position with the intratracheal cannula connected to a constant volume mechanical ventilator (Model SN-480-7; Shinano Co., Tokyo, Japan). A tidal volume of 10 ml per kg and a frequency of 60 breaths per minute were used. Transpulmonary pressure was measured with a pressure transducer (Model TP-603T; 50 c m H 2 0 ; Nihon Koden Co., Tokyo, Japan), with one side attached to a catheter inserted into the right pleural cavity and the other side attached to a catheter connected to a side port of the intratracheal cannula. The ventilatory circuit had a total volume of 18 ml. Airflow was measured with a pneumotachograph (Model TU-241T; Nihon Koden Co., Tokyo, Japan) connected to a transducer (Model TP-602T; 5 cm H 2 0 ; Nihon Koden Co., Tokyo, Japan). All signals were recorded on a 4-channel recorder (WT-645 GC; Nihon Koden Co., Tokyo, Japan). Lung resistance (R L) was calculated as described previously (Von Neergaard and Wirz, 1927).
2.2. Protocol I m m a t u r e or adult guinea pigs were divided into six groups each (total 12 groups) in order to compare the effects of serially diluted histamine (5, 15, 30 and 50 /xg/kg), 1 # g / k g substance P or their vehicle (0.9% NaC1) on airway microvascular leakage and increases in lung resistance for 6 min after administration. Ten minutes after connection to the ventilator, Evans Blue dye (20 m g / k g ) was administered i.v. One minute later, histamine was administered i.v. R L, transpulmonary pressure and mean blood pressure were recorded every
minute. Five minutes after drug administration, the animals were hyperinflated with twice the tidal volume by manually blocking the outflow of the ventilator, and 1 min later disconnected from the ventilator. We defined R L at 6 min as 'recovery R L' (L6Wall et al., 1990; Tokuyama et al., 1991a, b, c), because hyperinflation would be expected to diminish airway smooth muscle contraction and abolish airflow obstruction induced by atelectasis.
2.3. Determination of plasma extrat,asation After disconnection from the ventilator, the thoracic cavity was opened, and a cannula was inserted into the aorta through the left ventricle. Animals were perfused with 0.9% NaCI at a pressure of 100 mm Hg, to remove intravascular dye. The lungs were removed, and the lung parenchyma carefully scraped off. The trachea, main bronchi and intrapulmonary airways were separated from each other, and the intrapulmonary airways were divided into two equal portions lengthwise, arbitrarily named proximal and distal. All tissues were weighed wet. Evans Blue dye was extracted in 2 ml of formamide at 40°C for 24 h and measured in a spect r o p h o t o m e t e r (UV-150-02; Shimazu Co., Kyoto, Japan) at 620 nm. The extracted Evans Blue dye was quantified by interpolation on a standard curve of dye concentrations in the range of 0.5-10 ~zg/ml, and expressed as ng d y e / m g tissue. Evans Blue dye extravasation has been shown previously to correlate with the extravasation of radiolabelled albumin in guinea pig airways (Rogers et al., 1989).
2. 4. Drugs and chemicals The following drugs and chemicals were used: urethane and histamine (Kanto Chemical Co., Tokyo, Japan); Evans Blue dye (Aldrich Chemical Co., Milwaukee, USA); 0.9% NaCI (Fuyo Chemical Co., Osaka, Japan); substance P (Peninsula, USA).
2.5. Data analysis Data are reported as means_+ S.E.M. A M a n n Whitney U-test was used to test for significant differences between individual groups. A P value less than 0.05 was considered significant.
3. Results
3.1. Comparison of lung resistance Baseline R L w a s significantly higher in immature animals than in adults in all of the groups studied (fig. 1). In both immature and adult animals, histamine
53 TRACHEA
BASELINE
~oo~~z~
~
6O 40
~
E
t
g
"i"E u v
~
MAXIMAL RESPONSE
0L
E
Control
5
15
30 Histamine [#g,/kg]
50
MAIN BRONCHI
.~
8O 6O
--
~. I°°I ~
0
1
Substance P (#g/kgl
Fig. 1. Changes in lung resistance after i.v. histamine in immature and adult guinea pigs. Baseline (upper panel) and maximum increase in Rt. (lower panel) after control (0.9% NaCI), 5, 15, 30 and 50 /zg/kg histamine and 1 / z g / k g substance P in immature ( I ; n = 5, 5, 6, 5, 5 and 4, respectively) and adult guinea pigs ( [] ; n = 6, 7, 10, 6, 6 and 5, respectively) are shown. * P < 0.05 and ** P < 0.01 when comparing immature and adult animals receiving equal doses of histamine or substance P. + P < 0.05 and ++ P < 0.01 when compared with control animals of the same age.
40
~ m ~> to
PROXIMAL-IPA
80 60 40
20
~
o
DISTAL-IPA
m
~
0
caused a dose-dependent increase in R L, with the maximal response occurring within seconds after administration. The minimum dose of histamine to induce a significant increase in R e was 1 5 / z g / k g in both age groups. Substance P, 1 /zg/kg, did not cause a significant increase in R e compared to vehicle in either age group. The maximal response to 15, 30 and 50 / z g / k g histamine was greater in adults than in immature animals, with a significant difference at 5 0 / z g / k g histamine (fig. 1). Histamine, 50 /zg/kg, caused a marked fall in blood pressure and death in adults. In immature animals, it induced a higher value of the recovery R e (0.72 + 0.03) after histamine compared to the baseline R L (0.60 _+ 0.03), although this was not significant (0.05 < P < 0.1). In animals receiving 5, 15 or 30 / z g / k g histamine or its vehicle, recovery R g returned to baseline levels in both immature and adult animals.
3.2. Comparison of extraL:asation of Et,ans Blue dye At all airway levels, no significant difference was found in the extravasation of Evans Blue dye after vehicle (0.9% NaCI) between the immature and adult animals (fig. 2). Histamine caused a dose-dependent increase in the amount of dye exuded in both groups of animals. The minimum concentrations to induce significant extravasation of dye were 5 / z g / k g at trachea, 15 / z g / k g at main bronchi and proximal and distal intrapulmonary airways in immature animals, and 15 # g / k g at all airway levels in adults. We could not compare the degree of extravasated dye after 50 / x g / k g histamine, because this dose of histamine caused a marked fall in
i]
t
5
Control
15
30
Histamine [ ug/kg)
1
Substance P [,ug/kg)
Fig. 2. Airway microvascular leakage after different dose of histamine. Extravasation of Evans Blue dye at different airway levels after control (0.9% NaCI), 5, 15 and 30 /zg/kg histamine and 1 /zg/kg substance P in immature ( I ) and adult guinea pigs (12) are shown. Extravasation of dye in animals receiving 5 0 / x g / k g histamine could not be compared because many animals died, especially adult animals. * P < 0.05 and ** P < 0.01 when comparing immature and adult animals receiving equal doses of histamine or substance P. • P < 0.05 and ** P < 0.01 when compared with control animals of the same age. Numbers in columns show numbers of experimental animals in each group. IPA: intrapulmonary airways.
blood pressure and death trachea or main bronchi, differences in the amount mature and adult animals
in most adult animals. In there were no significant of dye exuded between imfor matched doses of his-
TABLE 1 Percent change in mean systemic blood pressure after i.v. administration of histamine in immature and adult guinea pigs. Values are means + S.E.M.; a p < 0.05 compared to control animals of the same age. No significant difference was found in the decrease in systemic blood pressure between immature and adult animals after the same doses of histamine. ( - ) : impossible to evaluate. Animals
Control (0.9% NaCI) Histamine 5 (/zg/kg) 15 30 50
Immature
Adult
1.8-+ 1.0 - 1.3 + 4.3 - 15.5 _+1.9 " - 17.2_+3.0 a - 17.6_+3.1 "
1.4_+2.0 -2.6_+2.6 - 11.4_+4.7 a -11.5_+5.1 a
(-)
54 tamine. In contrast, in proximal and distal intrapulmonary airways, there was significantly more dye exuded in immature animals after 15 or 30 /.tg/kg histamine than in adult animals. Substance P caused a significantly greater increase in dye extravasation at all airway levels than vehicle (0.9% NaCI). No significant differences in the amount of dye was observed at any airway levels between immature and adult animals after substance P (fig. 2).
3.3. Changes in blood pressure I.v. histamine caused a dose-dependent fall in systemic blood pressure (table 1). At 5, 15 and 30 p.g/kg, there were no significant differences in the decrease in blood pressure between immature and adult animals receiving corresponding doses of histamine. Histamine, 50/.tg/kg, induced a marked fall in blood pressure and death in adult animals. Substance P caused a marked fall in systemic blood pressure in both immature and adult animals ( - 5 9 . 5 + 3.0 and - 3 7 . 0 _+ 9.7% compared to the baseline levels, respectively), although there was no age-related difference.
4. Discussion
We have shown that immature guinea pigs required a larger dose of histamine than adults to achieve a similar degree of bronchoconstriction. In contrast, the degree of extravasation of Evans Blue dye after histamine was significantly greater in immature airways, especially in the periphery, while this was not the case after substance P. In both immature and adult animals, histamine caused a dose-dependent increase in RL, with maximal responses occurring within seconds after drug administration. Although the minimum dose to cause a significant increase in R L was not different between the two groups (15 /.tg/kg), the maximal response in R L was greater in adults, with a significant difference at 50 / z g / k g histamine. As observed previously (Clerici et al., 1989a, b), baseline R L was significantly higher in immature animals, presenting smaller airway diameters in resistance airways than in adults. A decrease in initial airway diameters has been shown to cause a greater increase in airflow resistance to stimuli, because resistance is inversely proportional to the fourth power of the airway radius (Barnes et al., 1988a). Therefore, our results suggest that smooth muscle in immature airways is less reactive to histamine, confirming previous observations in guinea pig (Clerici et al., 1989a), swine (Murphy et al., 1991) or sheep airways (Sauder et al., 1986). The lack of a vagal component in immature animals (Clerici et al., 1989a) and changes in the density or coupling of histamine H l receptor or in the
distribution of the two different histamine receptor subtypes (Ahmed et al., 1980) have been suggested to explain this age-related difference in response to histamine. Another possible explanation for this age-related difference is a decrease in the quantity of airway smooth muscle in immature airways, as has been described in human airways (Matsuba and Thurlbeck, 1972). In the present study, a concentration of substance P of 1 ~ g / k g was insufficient to cause a significant increase in R L in both age groups although it increased the extravasation of Evans Blue dye, confirming previous observations that substance P is much more potent in inducing airway microvascular leakage than in causing airway smooth muscle contraction (Rogers et al., 1988). In both age groups studied, histamine caused a dose-dependent increase in airway microvascular leakage at all airway levels. We could not compare the degree of extravasation after 50 /~g/kg histamine, because at this dose histamine caused a marked fall in blood pressure and death in adult animals. In contrast to trachea or main bronchi, where no significant difference was observed, a significantly greater dye extravasation was seen in most peripheral airways of immature animals after 15 or 30 g g / k g histamine. These results indicate that histamine induces a greater degree of microvascular leakage in immature airways, especially in peripheral airways. We do not know the mechanism of this airway level-dependent difference in microvascular leakage. Age-related changes in blood flow in peripheral airways might be a reason for this difference, because blood flow could be a major determinant in the degree of microvascular leakage (Persson, 1987). In contrast to histamine, substance P induced a greater extravasation of dye in adults in trachea and main bronchi, but caused no difference between adults and immatures in intrapulmonary airways. This result suggest that factors other than changes in blood flow with maturation might underlie an age-related difference in histamine-induced airway microvascular leakage, especially in peripheral airways. Changes in the density of histamine receptors or in the distribution of two different histamine receptor subtypes located in the microvenules might also contribute to this age-related difference. It has been shown that part of the plasma extravasation induced by histamine is mediated via sensory nerves (Lundberg and Saria, 1983). Therefore, the development of sensory nerves might be related to this difference. Murphy et al. (1991) have shown that, in young swine, i.v. histamine causes a significantly greater narrowing of airway diameter at peripheral airways, but not at central airways, than in adult animals. Airway edema resulting from airway microvascular leakage could be one of the contributory factors for this airway level-dependent difference in airway narrowing.
55
We have previously shown that histamine, when given by aerosol, causes a rapid increase in R L, peaking within seconds, followed by a partial recovery within minutes, with a prolonged elevation in R e which persists after hyperinflation of the lung (Tokuyama et al., 1991a, b, c). Although the initial increase is likely to reflect airway smooth muscle contraction in view of its rapid time course, we speculated that the more sustained phase was related, at least in part, to airway wall edema resulting from airway microvascular leakage, because the amount of dye exuded was significantly correlated with R e after hyperinflation (recovery R L) (L6tvall et al., 1990; Tokuyama et al., 1991a, b, c). In the present study, i.v. histamine caused a rapid increase in R t, which peaked within seconds, similar to the result obtained with aerosol administration. However, recovery R e in most animals returned to baseline levels. The difference in recovery R L between our previous observation (Tokuyama et al., 1991a, b, c) and present results may be due to the different routes of administration. Various pathogenic effects of histamine have been shown including anaphylaxis (Barnes et al., 1988). As shown in animals receiving 50 ~ g / k g histamine, i.v. administration might cause more severe cardiovascular responses than by aerosol administration, which would limit a large increase in R e or airway microvascular leakage. Indeed, the peak increase in R L was lower than in our previous study in which histamine was administered by aerosol (Tokuyama et al., 1991a, b, c). Histamine, as well as substance P, induced a significant decrease in systemic blood pressure in both immature and adult animals. Because changes in systemic blood flow may also affect the degree of microvascular leakage (Persson, 1987), changes in systemic blood pressure might be a cause of the age-related difference in the amount of dye exuded after histamine. However, this is unlikely, because no significant difference in changes in systemic blood pressure was observed between immature and adult animals receiving the same doses of histamine or substance P. We have shown that, in immature guinea pigs, the degree of airway microvascular leakage caused by histamine is greater, especially in the peripheral airways, while airway smooth muscle is less reactive to histamine than in adult guinea pigs. We do not know whether similar changes during maturation operate in human airways. Histamine has been shown to be a mediator of airway inflammation, including allergic inflammation (Barnes et al., 1988b). Therefore, if this were the case in man, airway edema or mucus plugs resulting from airway microvascular leakage might be greater in airways of young children than in adults during an asthmatic attack caused by several stimuli such as allergens, in spite of a lower responsiveness of the airway smooth muscle.
Acknowledgement We would like to thank Dr. Tetsuhiro Kubota, Kissei Pharmaceutical Company, Matsumoto, Japan, for his support of our experiments.
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activating factor: Role of airway microvascular leakage, Am. Rev. Respir. Dis. 143, 1345. Tokuyama, K., J.O. L6tvall, A. Morikawa, P.J. Barnes and K.F. Chung, 1991c, Role of thromboxane A 2 in airway microvascular leakage induced by inhaled platelet-activating factor, J. Appl. Physiol. 71, 1729. Von Neergaard, K. and K. Wirz, 1927, Die Messung der Stromungswiderstande in den Atemwegen des Menschen, insbesondere bei Asthma und Emphysem, Z. Klin. Med. 105, 51.
