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(Chest. 1999;116:1365-1368.)
© 1999 American College of Chest Physicians

Hydrogen Peroxide in the Lung Parenchyma Stimulates Vagally Mediated Phrenic Activity*

Galia K. Soukhova, PhD{dagger}; Muhammad Ahmed, MD; Eugene C. Fletcher, MD, FCCP and Jerry Yu, MD, PhD

* From the Pulmonary Division, Department of Medicine, University of Louisville, Louisville, KY. {dagger} Currently at the Pediatric Medical Academy of Russia, St. Petersburg, Russia.

Correspondence to: Jerry Yu, MD, PhD, Pulmonary Division, Department of Medicine, University of Louisville, ACB-3, 530 S Jackson St, Louisville, KY 40292; e-mail: j0yu0001{at}gwise.louisville.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Study objective: To elucidate the mechanism of hyperpnea and tachypnea, which are the common findings in cardiopulmonary patients.

Rational: Recently, it was found that activating pulmonary afferents by directly injecting hypertonic saline solution into the lung periphery causes a vagally mediated neural hyperpnea and tachypnea, ie, the excitatory lung reflex. Since reactive oxygen species are released during a variety of pulmonary diseases, we examined whether hydrogen peroxide (H2O2), a common mediator in cardiopulmonary diseases, can initiate the same excitatory lung reflex.

Measurements and results: We recorded phrenic efferent activity in anesthetized, open chest, artificially ventilated rabbits as an index of respiratory drive and examined the respiratory responses to injections of H2O2 (10 µmol in 0.1 mL). The responses were compared with those to hypertonic saline solution (8.1%, 0.1 mL). H2O2 and hypertonic saline solution increased both the rate (mean [± SEM], 43 ± 8% and 61 ± 10%, respectively; n = 30; p = 0.001) and the amplitude of phrenic bursts (12 ± 2% and 20 ± 4%, respectively; n = 30; p = 0.033). These responses were abolished by bilateral vagotomy.

Conclusion: H2O2 can initiate the excitatory lung reflex. Therefore, mediator(s) released in pulmonary diseases could be one of the mechanisms causing hyperpnea and tachypnea.

Key Words: breathing control • pulmonary receptors • reflex • vagus nerve


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Hyperpnea and tachypnea are common findings in patients with cardiopulmonary diseases involving the lung parenchyma. Despite vigorous efforts to investigate the responsible factors, the mechanisms that produce hyperpnea and tachypnea have not been completely identified. In an animal model of lung diseases,1 2 vagal afferents are found to mediate an alteration of a breathing pattern.3 Recently, we observed an excitatory lung reflex during activation of pulmonary afferents by directly injecting 0.1 mL hypertonic saline solution into the lung periphery of a rabbit.4 The reflex response included neural hyperpnea and tachypnea. Therefore, this reflex may have an important role in cardiopulmonary diseases. On the other hand, increased levels of reactive oxygen species, such as superoxide, hydrogen peroxide, and hydroxyl free radicals, have been associated with a number of pulmonary diseases,5 6 7 especially when the diseases involve the lung parenchyma. For example, oxygen radicals are thought to be involved in hyperoxia, emphysema, bronchopulmonary dysplasia, ARDS, and acute lung injury (such as bleomycin toxicity). We hypothesize that reactive oxygen species may activate vagal afferents in the lung periphery, thereby evoking hyperpnea and tachypnea. In the present study, H2O2 was locally injected into the lung parenchyma while whole phrenic nerve discharge was measured as an index of respiratory drive. Activation of the excitatory lung reflex would suggest the possibility that H2O2 may be responsible for hyperpnea and tachypnea in cardiopulmonary diseases involving lung parenchyma.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
General
Experiments were conducted on 35 male New Zealand white rabbits (body weight, 2.0 to 2.6 kg). The detailed procedures were described in a previous report.4 In short, the rabbits were initially anaesthetized with IM ketamine, 37.5 mg/kg, and IM xylazine, 5 mg/kg, and surgical anesthesia was maintained by additional doses of IV sodium pentobarbital (10 mg). During the experiment, anesthesia was maintained by IV infusion of {alpha}-chloralose (1%) and urethane (10%) at 1.1 to 2.0 mL/h. The trachea was cannulated low in the neck, the chest was opened, and the lungs were ventilated with room air by a small animal ventilator (model 683; Harvard Apparatus; South Natick MA) in which the expiratory outlet was connected to 3 to 4 cm H2O of positive end-expiratory pressure. Airway pressure was monitored by a pressure transducer attached to a side arm of the tracheal tube. Tidal volume was set at 10 mL/kg body weight. Ventilatory frequency was adjusted to maintain a constant peak airway PCO2 of about 40 mm Hg (which may have been an underestimate of alveolar PCO2). CO2 level was periodically monitored by an infrared analyzer (LB-2; SensorMedics; Yorba Linda, CA). The machine was calibrated by known CO2 concentrations. The femoral artery was cannulated for BP monitoring. Phrenic activity, its time-averaged signals, airway pressure, and BP were recorded by a thermorecorder (Dash IV; Astro-Med; West Warwick, RI).

To determine whether H2O2 can activate pulmonary receptors and reflexly stimulate breathing, we monitored phrenic activity as an index of respiratory drive and injected 0.1 mL H2O2 in 0.9% NaCl directly into the lung parenchyma (5 to 7 mm under the surface) through a 30-gauge needle. H2O2 was diluted to the desired concentration with 0.9% NaCl. In 30 rabbits, the response was compared with that to 0.1 mL injection of 8.1% NaCl. The response to 0.1 mL 0.9% NaCl was also examined as a vehicle control.

To determine whether H2O2-stimulated breathing is mediated through hydroxyl free radicals, we measured the respiratory responses to H2O2, 10 µmol in 0.1 mL, in 11 rabbits before and after local injection of the hydroxyl free radical scavenger, dimethyl sulfoxide (DMSO; 5% in 0.2 mL).

Phrenic Nerve Recordings
The phrenic nerve from C6 (right or left) was separated from the surrounding tissue and transected. The central end of the nerve was de-sheathed and placed on a bipolar silver electrode, which was connected to a high impedance probe (HIP5; Grass Instrument Division; West Warwick, RI) and then to an amplifier (P511; Grass Instrument Division). Nerve activity was monitored by a loudspeaker. Both the raw nerve signal and its "integrated signal," ie, its moving time-averaged signals obtained by a leaky integrator (7P3D; Grass Instrument Division; time constant, 50 ms), were recorded.8 The amplitude and rate of phrenic bursts were examined in response to the local injection of H2O2.

Data Analysis
Data are presented as mean (± SEM). A paired Student’s t test was used to compare two groups of data from the same animals. A p value < 0.05 was considered as statistically significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Injection of H2O2 (in 0.1 mL 0.9% NaCl) caused neural hyperpnea and tachypnea (Fig 1 ), which were exhibited by increases in the amplitude and burst rate of the phrenic neurogram. The response pattern to H2O2 is similar to that evoked by a local injection of 8.1% NaCl. The most prominent response is neural tachypnea (Figs 1 , 2 ); in addition, the mean arterial BP increased by 5.5 ± 0.7 and 6.7 ± 0.6 mm Hg, respectively in response to the injections of H2O2 and 8.1% NaCl (n = 20; p < 0.01). There was no difference between the increases in BP. On the other hand, as we reported in a previous study,4 injecting 0.1 mL 0.9% NaCl did not alter the respiratory pattern (Fig 3 ).



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  		Figure 1. Similar respiratory responses to injection of 0.1 mL 8.1% NaCl (top panel) or 1 µmol H2O2 (bottom panel) into the periphery of the right lung in an anesthetized, open-chest rabbit receiving artificial ventilation. Traces from top to bottom: ENG, the electroneurogram of the left phrenic nerve; ENG (Integ.), the "integrated" (time-averaged) electroneurogram; Paw, airway pressure; and BP. Three event marks on the top denote the insertion of the needle, and the start and end of the injection. Note that the phrenic activity increased immediately. The breathing rate was still linked to the ventilator cycle. The phrenic nerve gave two bursts, although fused, per ventilator cycle in some cases. Also note that there was an increase in BP after the injections.

 


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  		Figure 2. A comparison of respiratory responses to local injections of hydrogen peroxide, 10 µmol; (open bar) and hypertonic saline solution (crossed bar; n = 30). The responses are expressed as the percent increase in phrenic amplitude or frequency (burst rate) from the baseline after the initiation of the excitatory lung reflex. *Denotes the difference (p < 0.05) in responses between the two agents.

 


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  		Figure 3. H2O2, 10 µmol in 0.1 mL 0.9% NaCl, (top panel) injected into the periphery of the right lung evoked the excitatory lung reflex but not the injection of 0.9% NaCl (bottom panel). See Figure 1 legend for abbreviations.

 
In order to determine whether the excitatory reflex can be repeatedly evoked at the same injection point, we measured the respiratory responses to an injection of H2O2, 10 µmol in 0.1 mL 0.9% NaCl, at an interval of every 30 min for 2.5 h (n = 9). It was found that the response can be repeated for six injections without a change in rate and amplitude of phrenic response (Fig 4 ).



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  		Figure 4. Percent increases in the phrenic burst rate after the injection of H2O2 (10 µmol in 0.1 mL; n = 9). H2O2 was injected at 30-min intervals for six injections at the same injection point. There is no statistical difference among the injections.

 
In 11 rabbits, we measured the respiratory responses to H2O2, 10 µmol in 0.1 mL, before and after a local injection of 5% DMSO (0.2 mL) and found that there was no significant difference between DMSO-treated and nontreated responses to H2O2. It is worth a notice that DMSO itself did not cause any changes in phrenic activity. H2O2 injection increased the rate of phrenic bursts by 41.7 ± 10% before DMSO and by 38.6 ± 10% after DMSO (p > 0.05). To further ensure that DMSO and H2O2 distribute to the same extent, we compared the responses of four rabbits to H2O2 (10 µmol) and to a mixture containing H2O2 (10 µmol) and 5% DMSO. Again, we found no difference between the two responses. In five separate rabbits, the respiratory responses to H2O2 were abolished after bilateral cervical vagotomy.


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
Hyperpnea, tachypnea, and dyspnea are common findings in many cardiopulmonary diseases. Recently, an excitatory, vagally mediated reflex in the lung (excitatory lung reflex) was identified in the rabbit.4 Injection of hypertonic saline solution (8.1%) directly into the lung parenchyma, in a volume (0.1 mL) that is a fraction of the deflated lung volume (40 mL), initiates the excitatory lung reflex. It increased phrenic activity, which was exhibited by an increased amplitude and rate of bursts without substantial effects on the cardiovascular system. This reflex may be important in the pathophysiologic process of several pulmonary diseases.4 It is interesting that a local injection of phenylbiguanide, a C fiber stimulant, did not evoke this reflex.9

Reactive oxygen species are known as mediators for many cardiopulmonary pathophysiologic pro-cesses.6 7 Therefore, they could be responsible for the common symptoms and signs of cardiopulmonary diseases. The aim of the present study was to determine whether reactive oxygen species, specifically H2O2, can initiate the excitatory lung reflex. Indeed, our results show that an injection of 10 µmol H2O2 stimulated breathing. Thus, the present results provide the first evidence to demonstrate that H2O2 can initiate the excitatory lung reflex, thereby causing neural hyperpnea and tachypnea. Since H2O2 is a common mediator released during many cardiopulmonary diseases, the present study lends support for the hypothesis that the excitatory lung reflex is responsible for hyperpnea and tachypnea in cardiopulmonary diseases.

Reactive oxygen species are believed to be responsible for many diseases in different organ systems.10 11 They are recognized as playing an important role in many pulmonary diseases.6 7 12 It has been suggested that hydroxyl free radicals activate pulmonary C fibers in dogs.13 Directly applying H2O2 to the surface of the heart initiates a cardiac reflex through the vagal afferents.14 H2O2 has also been shown to evoke a vagally mediated reflex when applied topically to the GI tract.15 Until now, there was no direct evidence to show that H2O2 could stimulate pulmonary afferents to cause reflex effects on breathing. By employing a local injection technique, we were able to directly deliver H2O2 to the vicinity of pulmonary receptors. Our data provide convincing evidence that H2O2 can evoke the excitatory lung reflex because 0.9% NaCl containing H2O2 produced neural hyperpnea and tachypnea, but pure 0.9% NaCl did not. The amount of H2O2 (10 µmol, and in some cases, even 1 µmol) used to evoke the excitatory lung reflex is comparable with the amount used in activating vagal afferents in the GI tract (44 µmol)15 or in the heart (3 µmol)14. It is likely that the concentration at the immediate vicinity of the receptor field is much lower than the concentration injected because there should be a concentration gradient from the injection point to the nerve endings. In addition, H2O2 is readily metabolized by catalase and glutathione enzymes, which are found in the lung.11 16 Therefore, the concentration of H2O2 diffused to the receptor field should be far less than that applied.

Our experiments were not designed to determine by which mechanism H2O2 activates pulmonary receptors. However, our data could be used to exclude some possibilities. H2O2 can cause cell death by lysis in several cell lines.12 However, our data do not prove this mechanism to be responsible. If this were the case, repeated injections of H2O2 at the same place would abolish or at least attenuate the response. It is possible that the activation of the excitatory lung reflex is due to the production of an hydroxyl free radical. However, some arguments can be used against it. First, H2O2 evoked the response within a few seconds. It is known that H2O2 takes time to convert to a hydroxyl free radical, especially under a circumstance in which there are no abundant transitional ions.17 Second, pretreatment with DMSO or mixing H2O2 with DMSO did not prevent the response to H2O2. However, it still could be argued that DMSO failed to protect the initiation of the excitatory lung reflex because H2O2 diffused to the area of the nerve ending where DMSO did not reach. On the other hand, DMSO is a small molecule, is lipid soluble, and therefore, should access the area where H2O2 diffused. However, at this point, we do not have direct evidence to accept or refute this argument.


   Conclusion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Our study provides the first evidence that H2O2, a common mediator released during many cardiopulmonary diseases, can initiate the excitatory lung reflex, thereby causing neural hyperpnea and tachypnea by activating pulmonary afferents.


   Footnotes
 
Abbreviation: DMSO = dimethyl sulfoxide

Dr. Yu is supported by the American Heart Association, the

Mid-America Research Consortium (#9806306), NIH (HL58727–01), and the American Lung Association (CI-018-N).

Received for publication February 16, 1999. Accepted for publication June 8, 1999.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

  1. Armstrong, DJ, Luck, JC, Martin, VM (1976) The effect of emboli upon intrapulmonary receptors in the cat. Respir Physiol 26,41-54[CrossRef][ISI][Medline]
  2. Guz, A, Trenchard, DW (1971) The role of non-myelinated vagal afferent fibers from the lungs in the genesis of tachypnoea in the rabbit. J Physiol 213,345-347[Abstract/Free Full Text]
  3. Coleridge, HM, Coleridge, JCG (1994) Pulmonary reflexes: neural mechanisms of pulmonary defense. Annu Rev Physiol 56,69-91[CrossRef][ISI][Medline]
  4. Yu, J, Zhang, JF, Fletcher, EC (1998) Stimulation of breathing by activation of pulmonary peripheral afferents in rabbits. J Appl Physiol 85,1485-1492[Abstract/Free Full Text]
  5. Lai, YL (1990) Oxygen radicals in capsaicin-induced bronchoconstriction. J Appl Physiol 68,568-573[Abstract/Free Full Text]
  6. Repine, JE, Heffner, JE (1997) Lung antioxidants. Crystal, RG West, JB Weibel, ER eds. The lung: scientific foundations 2nd ed. ,2259-2269 Lippincott-Raven Philadelphia, PA.
  7. Warren, JS, Ward, PA (1997) Consequences of oxidant injury. Crystal, RG West, JB Weibel, ER eds. The lung: scientific foundations 2nd ed. ,2279-2288 Lippincott-Raven Philadelphia, PA.
  8. Eldridge, FL (1975) Relationship between respiratory nerve and muscle activity and muscle force output. J Appl Physiol 39,567-574[Abstract/Free Full Text]
  9. Yu, J, Soukhova, GK, Fletcher, EC (1998) Unclassified pulmonary afferent maybe responsible for excitatory lung reflex [abstract]. FASEB J 12,A782
  10. Halliwell, B, Gutteridge, JMC, Cross, CE (1992) Free radicals, antioxidants, and human disease: where are we now? J Lab Clin Med 119,598-620[ISI][Medline]
  11. Zimmerman, JJ (1995) Oxyradical pathophysiology. Adv Pediatr 42,243-302[Medline]
  12. Schraufstatter, IU, Cochrane, CG (1997) Oxidants: types, sources, and mechanisms of injury. Crystal, RG West, JB Weibel, ER eds. The lung: scientific foundations 2nd ed. ,2251-2258 Lippincott-Raven Philadelphia, PA.
  13. Chen, HF, Lee, BP, Kou, YR (1997) Mechanisms of stimulation of vagal pulmonary C fibers by pulmonary air embolism in dogs. J Appl Physiol 82,765-771[Abstract/Free Full Text]
  14. Ustinova, EE, Schultz, HD (1994) Activation of cardiac vagal afferents by oxygen-derived free radicals in rats. Circ Res 74,895-903[Abstract/Free Full Text]
  15. Stahl, GL, Pan, HL, Longhurst, JC (1993) Activation of ischemia- and reperfusion-sensitive abdominal visceral C fiber afferents: role of hydrogen peroxide and hydroxyl radicals. Circ Res 72,1266-1275[Abstract/Free Full Text]
  16. Cantin, AM, North, SL, Hubbard, RC, et al (1987) Normal alveolar epithelial lining fluid contains high levels of glutathione. J Appl Physiol 63,152-157[Abstract/Free Full Text]
  17. Halliwell, B, Cross, CE, Vliet, A, et al (1996) The generation and activity of oxygen-derived species. Weir, EK Archer, SL Reeves, JT eds. Nitric oxide and radicals in the pulmonary vasculature ,21-38 Futura New York, NY.



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This Article
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