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Published in final edited form as: Respir Physiol Neurobiol. 2015 Apr 9;0:46–49. doi: 10.1016/j.resp.2015.04.002

Airway extravasation induced by increasing airway temperature in ovalbumin-sensitized rats

Chun-Chun Hsu 1, Reyno J Tapia 1, Lu-Yuan Lee 1,*
PMCID: PMC4437191  NIHMSID: NIHMS687236  PMID: 25864799

Abstract

This study was carried out to determine whether hyperventilation of humidified warm air (HWA) induced airway extravasation in ovalbumin (Ova)-sensitized rats. Our results showed: 1) After isocapnic hyperventilation with HWA for 2 min, tracheal temperature (Ttr) was increased to 40.3°C, and the Evans blue contents in major airways and lung tissue were elevated to 651% and 707%, respectively, of that after hyperventilation with humidified room air in Ova-sensitized rats; this striking effect of HWA was absent in control rats. 2) The HWA-induced increase in Evans blue content in sensitized rats was completely prevented by a pretreatment with either L-732138, a selective antagonist of neurokinin type 1 (NK-1) receptor, or formoterol, a selective agonist of β2 adrenoceptor. This study demonstrated that an increase in airway temperature induced protein extravasation in the major airways and lung tissue of sensitized rats, and an activation of the NK-1 receptor by tachykinins released from bronchopulmonary C-fiber nerve endings was primarily responsible.

Keywords: asthma, vagus, C-fiber, tachykinins, bronchoconstriction

1. Introduction

A recent study carried out in our lab reported that hyperventilation (40% of maximal voluntary ventilation for 4 min) of humidified warm air (HWA) triggered cough and bronchoconstriction in patients with mild and stable asthma, and activation of bronchopulmonary sensory nerves by HWA was believed to be responsible (Hayes et al., 2012). A follow-up study further demonstrated that an increase in airway temperature resulting from hyperventilation of HWA evoked a pronounced increase in airway resistance in ovalbumin (Ova)-sensitized Brown- Norway rats, and that endogenous tachykinins were the primary contributing factor (Hsu et al., 2013). Furthermore, the HWA-induced airway response was not prevented by atropine and sustained for >10 min (Hsu et al., 2013), suggesting that the effect was not generated by the reflex-mediated airway smooth muscle contraction in anesthetized rats.

Tachykinins, such as substance P (SP) and neurokinin (NK) A, are pro-inflammatory neuropeptides released from pulmonary C-fiber sensory nerve endings upon intense stimulation. These neuropeptides can trigger a number of neurogenic inflammatory responses in the airways, including protein extravasation, mucosal edema and bronchoconstriction, by activating NK type 1 (NK-1) and type 2 (NK-2) receptors expressed on a number of target cells in the airways, including endothelial cells in capillaries and venules, airway and vascular smooth muscles (Steinhoff et al., 2014). However, whether airway extravasation was evoked by the HWA challenge in Ova-sensitized rats is not known (Hsu et al., 2013).

This study was carried out to answer the following questions: 1) Does hyperventilation of HWA induce airway extravasation, and if so, is the effect augmented in Ova-sensitized rats? 2) Are tachykinins responsible for the HWA-evoked airway extravasation, and if so, what are the relative roles of NK-1 and NK-2 receptors in mediating these responses?

2. Materials and Methods

This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The study protocol was approved by the University of Kentucky Institutional Animal Care and Use Committee.

2.1 Animal Sensitization and Preparation

Adult male pathogen-free Brown-Norway rats (267.5 ± 3.3 g) were separated into control and sensitized groups. Sensitized rats received an initial intraperitoneal (ip) injection of a suspension containing 2 mg of Ova (Sigma-Aldrich, St. Louis, MO) in 1 ml of Imject Alum (Pierce Biotechnology, Rockford, IL) as adjuvant. Three days later, rats were exposed to 1.25% Ova aerosol for 15 min three times per week (M/W/F) for 3 weeks, following the protocol identical to that described in our previous study (Hsu et al., 2013). Control rats received an ip injection of Imject Alum and inhalation of aerosolized vehicle (isotonic saline). One day after the last Ova or saline exposure, rats were anesthetized with α-chloralose (100 mg/kg; Sigma-Aldrich) and urethane (500 mg/kg; Sigma-Aldrich), placed in a supine position and ventilated mechanically with a respirator (model 683; Harvard, South Natick, MA) via a short tracheal cannula inserted just below the larynx. Respiratory rate (f) was set at 60 breaths/min, and tidal volume (VT) at 6–7 ml/kg. Body temperature was maintained at 36°C by a heating blanket. A polyethylene catheter was inserted into the left femoral vein for intravenous (iv) bolus injections of drugs.

2.2 HWA Challenge

The method for the HWA challenge was described in details in a previous report (Hsu et al., 2013). Briefly, the outlet of the respirator inspiratory line was connected to an air stone that was immersed in isotonic saline contained in a bottle placed in a heated water bath. HWA was then delivered directly into the lung via the tracheal tube. Humidified room air (HRA) was delivered in the same manner except that the water bath was kept at the room temperature (~23°C). During both HWA and HRA challenges, minute ventilation was elevated to ~375% of the baseline (by increasing VT and f to 150% and 250% of their baselines, respectively) for 2 min; ~4.0% of CO2 was added to the inspired air to maintain the isocapnic condition during hyperventilation. A miniature temperature probe (model IT-18; Physitemp, Clifton, NJ) was inserted into the tracheal tube and positioned near the thoracic inlet to continuously measure the air temperature in the trachea before and during HWA and HRA challenges.

2.3 Measurement of Airway Extravasation

Evans blue dye (30 mg/kg iv; Sigma-Aldrich) was injected 5 min before the HWA or HRA challenge. Ten min after the HWA or HRA challenge, the thorax was opened. To remove blood and intravascular Evans blue dye, pulmonary circulation and systemic circulation were perfused with isotonic saline via cannulated pulmonary artery (pressure, 30 mmHg; volume, 25 ml) and aorta (pressure, 120 mmHg; volume, 200 ml), respectively. The whole lung including trachea was then removed, separated into major airways (trachea and major bronchi) and lung tissue (lung parenchyma and intrapulmonary airways), and weighted. The intensity of airway extravasation was quantified by measuring the extravasation amount of Evans blue dye (Evans et al., 1988) extracted from tissue in formamide (Sigma-Aldrich) by incubation at 40°C in water bath for 24 hr and measured by light absorbance (SpectraMax, M2; Molecular Devices, Sunnyvale, CA) at 620 nm. The Evans blue content in tissue was determined from a standard curve of the dye in the concentration range of 0.05–20 µg/ml, and expressed as ng Evans blue dye per mg wet tissue (ng/mg).

2.4 Experimental Protocols

Four series of experiments were carried out. Series 1 aimed to determine if airway extravasation was generated by the increase of tracheal temperature (Ttr) induced by the HWA challenge; and to compare the responses between control and Ova-sensitized rats. Both control and sensitized rats were divided into two groups (n=6 in each group) for HWA and HRA challenges. Series 2: To investigate the role of the endogenous tachykinins, the HWA-induced extravasation responses in Ova-sensitized rats were compared between a control group (pretreated with vehicle) and a group pretreated with a combination of L-732138 (6 mg/kg iv; Tocris, Ellisville, MO), a selective NK-1 antagonist, and SR-48968 (1 mg/kg iv; Sanofi Recherche, Montpellier, France), a selective NK-2 antagonist 20 min earlier. Series 3: To determine the relative contributions of NK-1 and NK-2 receptors, the HWA-induced extravasation responses were compared between two groups of Ova-sensitized rats pretreated with L-732138 alone and SR-48968 alone, respectively, 20 min earlier. Series 4: To study the role of β2 adrenoceptors, the HWA-induced extravasation responses were determined in Ova-sensitized rats pretreated with formoterol (10 µg/kg iv; Sigma-Aldrich), a selective β2 agonist, 60 min earlier.

2.5 Statistical Analysis

Data were compared using one-way or two-way analysis of variance (ANOVA), followed by a post hoc Fisher’s test. P values of < 0.05 were considered significant. Data are reported as means ± SE.

3. Results

A total of 49 rats were used in this study. Despite that the age was matched between the two groups, the average body weight of Ova-sensitized rats (259 ± 3 g; n = 37) was significantly lower than that of control rats (293 ± 4 g; n=12, P < 0.01). Hyperventilation of HWA elevated Ttr from 31.5 ± 0.5°C to 40.1 ± 0.3°C (n=6, P < 0.001) in control rats and from 31.8 ± 0.2°C to 40.3 ± 0.1°C (n=6, P < 0.001) in sensitized rats. Hyperventilation with HRA decreased Ttr (from 31.3 ± 0.4°C to 24.4 ± 0.4°C in control rats; from 31.4 ± 0.3°C to 24.4 ± 0.2°C in sensitized rats).

Series 1

The Evans blue content after HWA challenge was significantly higher than that after HRA in both major airways and lung tissue in Ova-sensitized rats, but not in control rats. In Ova-sensitized rats, Evans blue contents measured in major airways were 143.8 ± 34.4 ng/mg and 22.1 ± 5.3 ng/mg in the two groups receiving HWA and HRA challenges, respectively (P < 0.01, n=6; Fig. 1A). In lung tissue, the Evans blue contents were 29.7 ± 5.7 ng/mg and 4.2 ± 0.7 ng/mg in HWA and HRA groups, respectively (P < 0.01, n=6; Fig. 1B). In a sharp contrast, in either major airways or lung tissue, the Evans blue contents were not different between the two groups of control rats receiving HWA and HRA challenges (P > 0.05, n=6; Fig. 1).

Fig. 1. Comparison of the intensity of airway extravasation in response to hyperventilation with humidified warm air (HWA) and humidified room air (HRA) in control and ovalbumin (Ova)- sensitized rats.

Fig. 1

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Open bars, control groups; filled bars, Ova-sensitized groups. The intensity of protein extravasation was measured in ng of Evans blue content per mg of wet tissue weight (Evans blue content) in A: major airways (trachea and major bronchi); B: lung tissue (lung parenchyma and intrapulmonary airways). Data are means ± SE; 6 rats in each group. * Significant difference when corresponding data were compared between control and sensitized groups (P < 0.05) ; # significant difference when corresponding data were compared between HWA and HRA responses (P < 0.05).

Series 2

Pretreatment with a combination of L-732138 and SR-48968 completely abolished the increase in Evans blue content in both major airways and lung tissue induced by the HWA challenge in Ova-sensitized rats (Fig. 2). In major airways, the Evans blue contents after HWA were 118.7 ± 11.6 in the control (vehicle) group, and 17.8 ± 1.9 ng/mg in the group pretreated with a combination of L-732138 and SR-48968 (P < 0.01, n=5; Fig. 2A); in lung tissue, the Evans blue contents after HWA were 30.9 ± 6.3 and 5.8 ± 0.6 ng/mg in the control (vehicle) and treated (L-732138+SR-48968) groups, respectively (P < 0.01, n=5; Fig. 2B).

Fig. 2. Role of endogenous tachykinins and formoterol in the HWA-induced airway extravasation in five different groups of Ova-sensitized rats.

Fig. 2

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Open bars, responses to HWA challenge in the control (pretreated with vehicle) group; hatched bars, responses to HWA challenge after pretreatment with a combination of L-732138 and SR-48968 (L+SR); cross-hatched bars, responses to HWA challenge after pretreatment with L-732138, the selective NK-1 antagonist (L; 6 mg/kg iv), alone; filled bars, responses to HWA challenge after pretreatment with SR-48968, the selective NK-2 antagonist, alone (SR; 1 mg/kg iv); shaded bars, responses to HWA challenge after pretreatment with formoterol (10 µg/kg iv), the β2 agonist. See the legend of Figure 1 for further explanations. Data are means ± SE; 5 rats in each group. * Significantly different from the response to HWA challenge in the control (vehicle) group (P < 0.05).

Series 3

Pretreatment with L-732138 alone significantly attenuated the HWA-induced increase in Evans blue content to 20.9 ± 1.0 ng/mg (P < 0.01, n=5; Fig. 2A) in major airways, and to 3.8 ± 0.2 ng/mg (P < 0.01, n=5; Fig. 2B) in lung tissue in Ova-sensitized rats. In contrast, pretreatment with SR-48968 alone did not significantly change the HWA-induced increase in Evans blue content in major airways (120.0 ± 29.5 ng/mg; P > 0.05, n=5; Fig. 2A), but reduced it by 40% in the lung tissue (18.4 ± 2.0 ng/mg; P < 0.05, n=5; Fig. 2B).

Series 4

Pretreatment with formoterol completely abolished the HWA-induced increase in Evans blue content in Ova-sensitized rats: 15.7 ± 3.2 ng/mg (P < 0.01, n=5; Fig. 2A) in major airways, and 5.7 ± 0.5 ng/mg (P < 0.01, n=5; Fig. 2B) in lung tissue.

4. Discussion

Results from this study have demonstrated that an increase in Ttr to 40.3°C after hyperventilation of HWA for 2 min evoked a pronounced increase in the Evans blue content in both major airways and lung tissue in Ova-sensitized rats (Fig. 1). In contrast, the same increase in Ttr generated by the HWA challenge did not induce any significant increase in Evans blue content in control rats (Fig. 1). Furthermore, the airway extravasation induced by HWA challenge in sensitized rats was completely prevented by pretreatment with a combination of selective NK-1 and NK-2 antagonists, clearly indicating the critical role of endogenously released tachykinins in this effect (Fig. 2). More importantly, our results further demonstrated a primary role of NK-1 receptors in this action of tachykinins since pretreatment with the NK-1 antagonist alone abolished the airway extravasation response to HWA challenge (Fig. 2).

Tachykinins consist of a family of structurally-related small peptides that share a common carboxyl terminal amino acid sequence. In the respiratory tract, two major types of tachykinins have been identified: SP and NKA; they are synthesized mainly in the cell bodies of bronchopulmonary C-fiber afferent nerves, co-localized and co-released form the sensory terminals upon stimulation. SP and NKA have preferential affinities for NK-1 and NK-2 receptors, respectively (Steinhoff et al., 2014). Activation of the NK-1 receptor by SP causes an increase in vascular permeability to plasma macromolecules and adhesion of leukocytes to the vascular endothelium; this effect takes place primarily at the postcapillary venules (Baluk et al., 1998; McDonald, 1988). Activation of the NK-1 receptor also causes smooth muscle relaxation in the arterioles and induces vasodilatation in microvessels. Together, they can cause protein extravasation, mucosal swelling and edema in the airways, which in turn can lead to a reduced airway luminal caliber and an increase in airway resistance.

Several factors are probably involved in the extravasation response observed in this study. One possible mechanism is that chronic airway inflammation may have increased pulmonary Cfiber sensitivity to the HWA challenge in Ova-sensitized rats, which is in agreement with our recent observation in patients with asthma; the same HWA hyperventilation challenge evoked a more intense response of cough and reflex bronchoconstriction in asthmatics than in healthy individuals (Hayes et al., 2012). Indeed, a preliminary study carried out in our lab has shown that the same increase in Ttr induced by the HWA challenge evoked a greater intensity of discharge of bronchopulmonatry C-fiber afferents in Ova-sensitized rats (Lin and Lee, 2013), which presumably caused a larger quantity of tachykinins to be released from these sensory endings. In addition, previous investigators have demonstrated that Ova-sensitization increased the amount of tachykinins synthesized and released in guinea pig airways (Fischer et al., 1996). Chronic allergic inflammation also induced a phenotypic switch in the tachykinergic innervation of airways (Myers et al., 2002). Taken together, all these mechanisms may have contributed, to some extent, to the pronounced protein extravasation response to the HWA challenge in the Ova-sensitized airways.

Our results showed that the degree of the HWA-evoked extravasation in Ova-sensitized rats was more pronounced in the trachea and major bronchi than that in the lung tissue (Fig. 1). This difference was probably related to an expected greater increase in temperature generated by the HWA inhalation challenge in the major airways than that in the peripheral airways and distal regions of the lung. We should also point out that the Evans blue content measured in the lung tissue included that from both lung parenchyma and intrapulmonary airways in this study.

A recent study of our lab has shown that the HWA-induced increase in airway resistance in Ova-sensitized Brown-Norway rats was effectively prevented by a pretreatment with formoterol (Hsu et al., 2013). This observation led us to suggest a possible involvement of airway smooth muscle contraction in the HWA-evoked increase in airway resistance because of the potent dilating effect of formoterol on airway smooth muscles. However, selective β2 agonists such as formoterol are also known to exert other potent therapeutic effects during acute lung injury, including edema clearance and anti-inflammatory effects (Groshaus et al., 2004). Indeed, previous investigators have demonstrated that activation of β2 adrenoceptors expressed in the airway vascular endothelial cells increased the intracellular cAMP level in these cells, which resulted in a reduction of the number but not the size of endothelial gaps in postcapillary venules, and diminished the protein extravasation (Baluk and McDonald, 1994). These findings may, therefore, provide the explanation for the effective blocking effect of formoterol on the HWA-evoked airway extravasation in Ova-sensitized rats. Although both formoterol and L-732138 appeared to act on the postcapillary venule endothelial cells, whether their actions are mediated through the same cellular mechanisms is not known since activation of NK-1 receptors can also initiate other G protein-mediated signaling pathways such as activations of phospholipases Cβ and A2, in addition to adenylyl cyclase (Steinhoff et al., 2014).

In summary, an increase in airway temperature within the physiological range induced protein extravasation in major airways and lung tissue in Ova-sensitized rats, but not in control rats. This response was mediated through an activation of the NK-1 receptor by tachykinins released from bronchopulmonary C-fiber nerve endings.

Highlights.

  1. An increase in airway temperature after hyperventilation of humidified warm air (HWA) for 2 min induced protein extravasation in the airways of ovalbumin-sensitized rats, but not in control rats.

  2. Pretreatment with neurokinin (NK)-1 antagonist abolished the airway extravasation response to HWA, indicating the primary action of endogenous tachykinins on NK-1 receptors.

  3. The HWA-induced airway extravasation was also completely prevented by formoterol, and the effect was probably caused by activation of β2 adrenoceptors expressed in airway vascular endothelial cells.

Acknowledgements

This study was supported in part by US Department of Defense DMRDP/USAMRMC/TATRC Award W81XWH-10-2-0189, NIH National Heart, Lung and Blood Institute grant HL-96914 and National Center for Advancing Translational Sciences grant UL1TR0000117.

Footnotes

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References

  1. Baluk P, Bolton P, Hirata A, Thurston G, McDonald DM. Endothelial gaps and adherent leukocytes in allergen-induced early- and late-phase plasma leakage in rat airways. The American journal of pathology. 1998;152:1463–1476. [PMC free article] [PubMed] [Google Scholar]
  2. Baluk P, McDonald DM. The beta 2-adrenergic receptor agonist formoterol reduces microvascular leakage by inhibiting endothelial gap formation. The American journal of physiology. 1994;266:L461–L468. doi: 10.1152/ajplung.1994.266.4.L461. [DOI] [PubMed] [Google Scholar]
  3. Evans TW, Rogers DF, Aursudkij B, Chung KF, Barnes PJ. Inflammatory mediators involved in antigen-induced airway microvascular leakage in guinea pigs. The American review of respiratory disease. 1988;138:395–399. doi: 10.1164/ajrccm/138.2.395. [DOI] [PubMed] [Google Scholar]
  4. Fischer A, McGregor GP, Saria A, Philippin B, Kummer W. Induction of tachykinin gene and peptide expression in guinea pig nodose primary afferent neurons by allergic airway inflammation. The Journal of clinical investigation. 1996;98:2284–2291. doi: 10.1172/JCI119039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Groshaus HE, Manocha S, Walley KR, Russell JA. Mechanisms of beta-receptor stimulation-induced improvement of acute lung injury and pulmonary edema. Critical care (London, England) 2004;8:234–242. doi: 10.1186/cc2875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hayes D, Jr, Collins PB, Khosravi M, Lin RL, Lee LY. Bronchoconstriction triggered by breathing hot humid air in patients with asthma: role of cholinergic reflex. American journal of respiratory and critical care medicine. 2012;185:1190–1196. doi: 10.1164/rccm.201201-0088OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hsu CC, Lin RL, Lin YS, Lee LY. Bronchoconstriction induced by increasing airway temperature in ovalbumin-sensitized rats: role of tachykinins. Journal of applied physiology (Bethesda, Md.: 1985) 2013;115:688–696. doi: 10.1152/japplphysiol.00491.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lin YJ, Lee LY. Hypersensitivity of bronchopulmonary C-fibers induced by an increase in airway temperature in ovalbumin (Ova)-sensitized Brown Norway rats (Abstract) The FASEB Journal. 2013 27,930.19. [Google Scholar]
  9. McDonald DM. Neurogenic inflammation in the rat trachea. I. Changes in venules, leucocytes and epithelial cells. Journal of neurocytology. 1988;17:583–603. doi: 10.1007/BF01260988. [DOI] [PubMed] [Google Scholar]
  10. Myers AC, Kajekar R, Undem BJ. Allergic inflammation-induced neuropeptide production in rapidly adapting afferent nerves in guinea pig airways. American journal of physiology. Lung cellular and molecular physiology. 2002;282:L775–L781. doi: 10.1152/ajplung.00353.2001. [DOI] [PubMed] [Google Scholar]
  11. Steinhoff MS, von Mentzer B, Geppetti P, Pothoulakis C, Bunnett NW. Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease. Physiological reviews. 2014;94:265–301. doi: 10.1152/physrev.00031.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]

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