Airway cooling and mucosal injury during cold weather exercise.

In human subjects that exercise strenuously in cold weather, there is evidence that hyperventilation with cold air leads to peripheral airway cooling, desiccation and mucosal injury. Our hypothesis was that hyperventilation with cold air can result in penetration of unconditioned air (air that is not completely warmed and humidified) into the peripheral airways of exercising horses, resulting in peripheral airway mucosal injury. To test this hypothesis, a thermister-tipped catheter was inserted through the midcervical trachea and advanced into a sublobar bronchus in three horses that cantered on a treadmill at 6.6 m/s while breathing cold (5 degrees C) air. The mean (+/- s.e.) intra-airway temperature during cantering was 33.3 +/- 0.4 degrees C, a value comparable to the bronchial lumen temperatures measured in man during maximal exercise while breathing subfreezing dry air. In a second experiment, 6 fit Thoroughbred racehorses with satisfactory performance were used to determine whether strenuous exercise in cold conditions can produce airway injury. Horses were assigned to Exercise (E) or Control (C) groups in a random crossover design. Samples of bronchoalveolar lavage fluid (BALF) in the E treatment were recovered within 30 min of galloping exercise in 4 degrees C, 100% relative humidity (E), while in C BALF samples were obtained when the horses had not performed any exercise for at least 48 h prior. Ciliated epithelial cells in BALF were higher in E than in the C treatment. Similar results have been found in human athletes and laboratory animal models of cold weather exercise. These results support the hypothesis that, similar to man, horses that exercise in cold weather experience peripheral airway mucosal injury due to the penetration of unconditioned air. Furthermore, these results suggest that airway cooling and desiccation may be a factor in airway inflammation commonly found in equine athletes.

Repeated hyperventilation causes peripheral airways inflammation, hyperreactivity, and impaired bronchodilation in dogs.

Winter athletes have an increased incidence of asthma, suggesting that repetitive hyperventilation with cold air may predispose individuals to airways disease. We used a canine model of exercise-induced hyperpnea to examine the effects of repeated hyperventilation with cool, dry air (i.e., dry air challenge [DAC]) on peripheral airway resistance (Rp), reactivity, and inflammation. Specific bronchi were exposed to a single DAC on five consecutive days. Rp and Delta Rp to aerosolized histamine, intravenous histamine, or hypocapnia were measured daily. Bronchoalveolar lavage fluid (BALF) was obtained on the fifth day. Rp increased from 0.70 +/- 0.08 to 1.13 +/- 0.22 cm H(2)O/ml/s (n = 25) 24 h after the first DAC, rose to 1.49 +/- 0.24 cm H(2)O/ml/s by Day 3, and remained elevated throughout the remainder of the protocol. Repeated DAC increased reactivity to hypocapnia and intravenous histamine. Intravenous salbutamol failed to reduce Rp as effectively in challenged airways (111% of Day 1 baseline) as in naive airways (54% of baseline). Repeated DAC caused increased BALF neutrophils, eosinophils, and sulfidopeptide leukotrienes. We conclude that repeated DAC causes peripheral airways inflammation, obstruction, hyperreactivity, and impaired beta-agonist-induced relaxation. This suggests that other mechanisms in addition to increased smooth muscle tone may contribute to the development of repetitive hyperventilation-induced bronchial obstruction and hyperreactivity.

Hypertonic saline aerosol increases airway reactivity in the canine lung periphery.

Hyperventilation with dry air increases airway surface fluid (ASF) osmolality and causes acute mucosal injury, leukocyte infiltration, and delayed airway obstruction and hyperreactivity in canine peripheral airways. The purpose of this study was to determine whether ASF hypertonicity per se can account for these hyperventilation-associated effects. We first measured ASF osmolality before and after normal (NSC) and hypertonic (HSC) saline aerosol challenges to document the magnitude of hypertonicity produced by these stimuli. We then measured canine peripheral airway resistance and reactivity to hypocapnia and aerosolized histamine before and after NSC and HSC. Cells and eicosanoid mediators recovered in bronchoalveolar lavage fluid at 5 and 24 h after NSC and HSC were examined. We found that HSC but not NSC caused acute ASF hyperosmolality, increased mediator release, and delayed airway hyperreactivity in the absence of mucosal injury and leukocyte infiltration. These observations suggest that ASF hyperosmolality contributes to the development of the late-phase response to hyperventilation and further suggest that hyperventilation-induced mucosal injury independently initiates leukocyte infiltration and late-phase airway obstruction.

Eicosanoid and muscarinic receptor blockade abolishes hyperventilation-induced bronchoconstriction.

This study was designed to test the hypothesis that hyperventilation-induced bronchoconstriction (HIB) results from the combined effects of prostanoid and leukotriene metabolism. A bronchoscope was used in anesthetized dogs to record peripheral airway resistance and HIB before and after combined treatment with inhibitors of cyclooxygenase (indomethacin) and 5-lipoxygenase (MK-0591). Bronchoalveolar lavage fluid (BALF) cells and mediators from hyperventilated and control airways were also measured. Pretreatment with MK-0591 and indomethacin significantly attenuated, but did not abolish, HIB. However, addition of atropine nearly eliminated the residual response. Blockade of eicosanoid metabolism markedly reduced the concentrations of eicosanoids recovered in BALF after hyperventilation. Positive correlations between posthyperventilation BALF prostanoid and epithelial cell concentrations are suggestive of mucosal injury-induced mediator production and release. We conclude that HIB is prevented in the presence of eicosanoid and muscarinic-receptor blockade and that both classes of eicosanoids contribute similarly to the development of HIB.

Heparin inhibits eicosanoid metabolism and hyperventilation-induced bronchoconstriction in dogs.

Inhalation of heparin, an anticoagulant, attenuates exercise- induced asthma (EIA) in human subjects. The purpose of this study was to determine if heparin inhibits hyperventilation-induced bronchoconstriction (HIB) in a canine model of EIA, and if its mode of action involves the inhibition of eicosanoid mediator production and release. We used a wedged bronchoscope technique to measure baseline peripheral airway resistance (Rp). We then performed either a 2-min or 5-min dry air challenge (DAC) by temporarily increasing from 200 to 2,000 ml/min the flow of 5% CO(2) in air used to ventilate a wedged sublobar segment. We compared HIB before and 60 min after aerosol treatment with either bacteriostatic water (BW) or heparin. We found that (1) heparin had no effect on baseline Rp, (2) BW did not alter the response to DAC, and (3) heparin reduced HIB by approximately 50-60%. On the basis of bronchoalveolar lavage fluid (BALF) cell analysis, heparin and BW caused acute infiltration of macrophages and eosinophils, and heparin increased the number of erythrocytes recovered immediately after DAC. Despite these acute inflammatory effects initiated prior to DAC, BALF mediator analyses revealed that pretreatment with heparin either attenuated or abolished hyperventilation-induced leukotriene, prostaglandin, and thromboxane release. Thus, our data provide direct evidence that inhaled heparin inhibits eicosanoid mediator production and release caused by hyperventilation with dry air, and significantly attenuates HIB.

Antioxidant transport modulates peripheral airway reactivity and inflammation during ozone exposure.

We examined the effects of ozone (O(3)) and endogenous antioxidant transport on canine peripheral airway function, central airway function, epithelial integrity, and inflammation. Dogs were either untreated or pretreated with probenecid (an anion-transport inhibitor) and exposed for 6 h to 0.2 parts/million O(3). Peripheral airway resistance (Rpa) and reactivity (DeltaRpa) were monitored in three sublobar locations before and after exposure to either air or O(3). Pulmonary resistance and transepithelial potential difference in trachea and bronchus were also recorded. Bronchoalveolar lavage fluid (BALF) was collected before, during, and after exposure. O(3) increased Rpa and DeltaRpa only in probenecid-treated dogs and in a location-dependent fashion. Pulmonary resistance and potential difference in bronchus increased after O(3) exposure regardless of treatment. O(3) markedly increased BALF neutrophils only in untreated dogs. With the exception of hexanal, O(3) did not alter any BALF constituent examined. Probenecid reduced BALF ascorbate, BALF protein, and plasma urate. We conclude that 1) a 6-h exposure to 0.2 parts/million O(3) represents a subthreshold stimulus in relation to its effects on peripheral airway function in dogs, 2) antioxidant transport contributes to the maintenance of normal airway tone and reactivity under conditions of oxidant stress, 3) O(3)-induced changes in Rpa and DeltaRpa are dependent on location, and 4) peripheral airway hyperreactivity and inflammation reflect independent responses to O(3) exposure. Finally, although anion transport mitigates the effect of O(3) on peripheral airway function, it contributes to the development of airway inflammation and may represent a possible target for anti-inflammatory prevention or therapy.

Mucosal injury and eicosanoid kinetics during hyperventilation-induced bronchoconstriction.

Bronchoalveolar lavage (BAL) of canine peripheral airways was performed at various times after hyperventilation, and BAL fluid (BALF) cell and mediator data were used to evaluate two hypotheses: 1) hyperventilation-induced mucosal injury stimulates mediator production, and 2) mucosal damage is correlated with the magnitude of hyperventilation-induced bronchoconstriction. We found that epithelial cells increased in BALF immediately after a 2- and a 5-min dry air challenge (DAC). Prostaglandins D(2) and F(2alpha) and thromboxane B(2) were unchanged immediately after a 2-min DAC but were significantly increased after a 5-min DAC. Leukotriene C(4), D(4), and E(4) did not increase until 5 min after DAC. Hyperventilation with warm moist air did not alter BALF cells or mediators and caused less airway obstruction that occurred earlier than DAC. BALF epithelial cells were correlated with mediator release, and mediator release and epithelial cells were correlated with hyperventilation-induced bronchoconstriction. These observations are consistent with the hypothesis that hyperventilation-induced mucosal damage initiates peripheral airway constriction via the release of biochemical mediators.

Repetitive hyperpnoea causes peripheral airway obstruction and eosinophilia.

Hyperpnoea of canine peripheral airways with dry air results in airway obstruction, mucosal damage, and inflammation. The purpose of this study was to evaluate the effect of repeated dry air challenge (DAC) on airway obstruction, reactivity and the development of airway inflammation in dogs. Canine peripheral airways received DAC (delivered under general anaesthesia through a bronchoscope) every 48 h for two weeks. Peripheral airway resistance and reactivity were measured prior to each DAC. After the final DAC, bronchoalveolar lavage fluid (BALF) cells and soluble mediators from challenged and control airways were measured. Repeated bronchoscopy had no effect on airway mechanics. Repeated DAC produced cumulative increases in peripheral airway resistance and peak obstructive response to DAC. The response to hypocapnia was also increased in airways receiving repeated DAC. However, when the response to agonists was expressed as a change from baseline, consistent significant increases were not observed. Repeated bronchoscopy produced insignificant changes in BALF cells and eicosanoid mediators. Repeated DAC produced marked eosinophilic inflammation and increased prostaglandins D2, E2, and F2alpha, as well as leukotrienes C4-E4. In conclusion, repeated dry air challenge in dogs in vivo causes persistent airway obstruction and inflammation not unlike that found in human asthma.

Hyperventilation with dry air increases airway surface fluid osmolality in canine peripheral airways.

Hyperventilation-induced bronchoconstriction (HIB) is a component of exercise-induced asthma (EIA) believed to result from the penetration of unconditioned air into the lung periphery. We used a canine model of EIA to examine the effect of hyperventilation on airway surface fluid (ASF) volume and osmolality, and to determine if the observed kinetics support the hypothesis that hyperventilation-induced changes in ASF osmolality initiate bronchoconstriction. Exposure of sublobar airways to dry air at baseline insufflation resulted in stable measurements of ASF volume, ASF osmolality, and peripheral airway resistance (Rp). Baseline insufflation of warm humidified air increased ASF volume, but did not alter ASF osmolality. Hyperventilation challenge with warm humidified air (WAC) increased Rp and ASF volume, but decreased ASF osmolality. Dry air challenge (DAC) increased Rp, ASF volume, and ASF osmolality. ASF osmolality during DAC was markedly higher when compared with posthyperventilation values. Post-DAC changes in (Delta) ASF volume and osmolality were poorly correlated with the development of HIB. In contrast to Delta ASF after DAC, Delta ASF osmolality during DAC was strongly correlated with HIB, and tended to be inversely related to Delta ASF volume. These observations are consistent with the hypothesis that changes in airway osmolality during hyperventilation initiate peripheral airway constriction.

Regional clearance of solute from peripheral airway epithelia: recovery after sublobar exposure to ozone.

The influence of local exposure to ozone (O3) on respiratory epithelial permeability of sublobar lung segments was studied by using aerosolized 99mTc-diethylenetriamine pentaacetic acid (DTPA; mol wt, 492). Two bronchoscopes were inserted through an endotracheal tube in anesthetized, mechanically ventilated, mixed breed dogs and were wedged into sublobar bronchi located in the right and left lower lobes, respectively. Segments were ventilated via the bronchoscope with 5% CO2 in air delivered at 200 ml/min, and an aerosol of 99mTc-DTPA was generated and delivered through the scope and into the sublobar segment over a 30-s period. Clearance of 99mTc-DTPA was measured simultaneously from right and left lower lung segments at baseline and 1, 7, and 14 days after a 6-h sublobar exposure to filtered air or 400 parts per billion O3. O3 treatment significantly decreased the clearance halftime (t50) of 99mTc-DTPA by 50% from the baseline mean of 32.3 to 16.0 min at 1 day postexposure. After 7 days of recovery, t50 was still reduced by 28. 8%; however, by 14 days postexposure, clearance of 99mTc-DTPA had recovered, and the t50 had a mean value of 30.0 min. 99mTc-DTPA clearance was not altered by exposure to filtered air, and t50 values were comparable to baseline at 1, 7, and 14 days postexposure. These results reveal that a single local exposure to O3 increases transepithelial clearance, but only for epithelia directly exposed to O3, and that 7-14 days of recovery are required before permeability to small-molecular-weight solutes returns to normal.

Retention of soluble 99mTc-DTPA in the human lung: 24-h postdeposition.

Clearance of low-molecular-weight solutes, e.g., radiolabeled chelate diethylenetriaminepentaacetate (DTPA), across epithelial surfaces of distal airways and the lung parenchyma is a broadly used technique to assess epithelial integrity. It has been generally assumed that clearance of solute follows a simple first-order process and that DTPA clearance through the respiratory epithelium and into blood and lymphatic channels is complete within a few hours. Using gamma-camera imaging and a radiolabeled aerosol of 99mTc-labeled DTPA, we observed in eight healthy subjects lung retention of radioisotope approximately 24 h postdeposition of the 99mTc-DTPA. Residual lung retention at the 24-h end point averaged 6.0 +/- 1.8 (SD)% of the amount of radioisotope initially deposited in the lung. This suggests that for normal healthy subjects a small amount of the 99mTc radioisotope, either in a dissociated or chelated form, is nonpermeable or slowly cleared from respiratory tisssues.

Hyperventilation-induced airway injury and vascular leakage in dogs: effects of alpha1-adrenergic agonists.

alpha1-Adrenergic agonists inhibit hyperventilation-induced bronchoconstriction (HIB) in dogs. We tested the hypothesis that alpha-agonists inhibit HIB by reducing bronchovascular leakage and edema that theoretically could cause airway obstruction. Peripheral airways were isolated by using a bronchoscope; pretreated with either methoxamine (Mx), norepinephrine (NE), or saline aerosol; and then exposed to a 2,000 ml/min dry-air challenge (DAC) for 2 min. Colloidal carbon was injected before DAC and used to quantify bronchovascular permeability. Mx-, NE-, and vehicle-treated airways were prepared for morphometric analysis within 1 h after DAC. Light microscopy revealed that the 2-min DAC produced minimal bronchovascular leakage and little epithelial damage. However, pretreatment with either Mx or NE significantly enhanced dry air-induced bronchovascular hyperpermeability and mucosal injury. The increased damage associated with these alpha1-agonists implicates a protective role for the bronchial circulation. The fact that alpha1-agonists inhibit HIB suggests that neither dry air-induced leakage nor injury directly contributes to the development of airway obstruction. In addition, our data suggest that alpha-agonists attenuate HIB in part by augmenting hyperventilation-induced bronchovascular leakage and by replacing airway water lost during a DAC.

Effect of furosemide on hyperpnea-induced airway obstruction, injury, and microvascular leakage.

Furosemide attenuates hyperpnea-induced airway obstruction (HIAO) in asthmatic subjects via unknown mechanism(s). We studied the effect of furosemide on dry air-induced bronchoconstriction, mucosal injury, and bronchovascular hyperpermeability in a canine model of exercise-induced asthma. Peripheral airway resistance (Rp) was recorded before and after a 2-min dry-air challenge (DAC) at 2,000 ml/min. After pretreatment with aerosolized saline containing 0.75% dimethyl sulfoxide, DAC increased Rp 72 +/- 11% (SE, n = 7) above baseline; aerosolized furosemide (10(-3) M) reduced this response by approximately 50 +/- 6% (P < 0.01). To assess bronchovascular permeability, colloidal carbon was injected (1 ml/kg i.v.) 1 min before DAC, and after 1 h, the vehicle- and furosemide-treated airways were prepared for morphometric analysis. Light microscopy confirmed previous studies showing that DAC damaged the airway epithelium and enhanced bronchovascular permeability. Furosemide did not inhibit dry air-induced mucosal injury or bronchovascular hyperpermeability and in fact tended to increase airway damage and vascular leakage. This positive trend toward enhanced bronchovascular permeability in DAC canine peripheral airways is consistent with the hypothesis that furosemide inhibits HIAO in part by enhancing microvascular leakage and thus counterbalancing the evaporative water loss that occurs during hyperpnea.

Eicosanoids modulate hyperpnea-induced bronchoconstriction in canine peripheral airways.

We examined the role of leukotrienes (LTs) in the development of dry air-induced bronchoconstriction (AIB) in canine peripheral airways. Airway reactivity to exogenous LTs was first tested by using an LTD4 aerosol challenge: peripheral airway resistance increased approximately 130 +/- 51% (n = 4) above baseline when compared with its vehicle control. AIB was then assessed by measuring peripheral airway resistance after, and airway wall temperature during, a dry air challenge (DAC). Treatment with a peptidoleukotriene biosynthesis inhibitor (MK-0591) attenuated AIB by approximately 65% without altering airway wall temperature. The fact that MK-0591 did not alter airway reactivity to aerosolized acetylcholine and completely inhibited Ca2+ ionophore-induced LTB4 generation in canine whole blood attests to the specificity of the drug. Treatment with MK-0591 did not affect the increased number of epithelial cells recovered in bronchoalveolar lavage fluid 5 min after DAC. Concentrations of LTs and other eicosanoids in bronchoalveolar lavage fluid from vehicle-treated DAC airways were increased above baseline values; only LTs were reduced by MK-0591. Before MK-0591, AIB was significantly correlated with the dry air-induced generation of LTC4, LTD4, and LTE4. After treatment with MK-0591, AIB was correlated with thromboxane B2, prostaglandin (PG) F2 alpha, and PGE2. We conclude that hyperpnea with dry air stimulates local production and release of LTs in canine bronchi and, alone with the generation of bronchoconstricting and bronchodilating PGs, plays a central role in the modulation of AIB.

Ozone-induced vagal reflex modulates airways reactivity in rabbits.

We examined the effects of ozone (O3) on central and peripheral airway reactivity and tracheal transepithelial potential difference (PD) in New Zealand white rabbits. Rabbits were exposed for 7 h to either room temperature-humidified filtered air (n = 7) or 0.2 ppm O3 in humidified room air (n = 5). Tracheal PD was recorded 3 h after exposure. Whole lung resistance (RL) and reactivity were partitioned into their central (RC) and peripheral (RP) components using a retrograde catheter and forced oscillation. Changes in RL, RC, and RP in response to NaCl (0.9%) and ACh (100 mM) aerosol challenges were measured before and after vagotomy. Exposure to O3 decreased tracheal PD from -29 +/- 0.6 mV in air-exposed rabbits to -15 +/- 2 mV in O3-exposed rabbits (p < or = 0.0001). Exposure to O3 did not alter RL, RC, or RP. However, the ACh-induced increase in RL in O3-exposed rabbits (140%) was twice that recorded in the air-exposed group (p < or = 0.01). While changes in RP dominated the whole lung response to ACh in air-exposed rabbits, changes in RC were most prominent in the O3-exposed group. Bilateral vagotomy did not alter airway reactivity in control rabbits but did enhance peripheral lung reactivity in O3-exposed rabbits. We conclude that exposure to 0.2 ppm O3 for 7 h affects tracheal epithelial function in rabbits and increases central airway reactivity via vagal mechanisms without altering baseline RL, RC, or RP.

Models and mechanisms of exercise-induced asthma.

Airflow-induced bronchoconstriction (AIB) in mammals can be broadly categorized as either vagal-dependent or vagal-independent. Among mammals, rabbits and cats belong to the former and guinea-pigs belong to the latter categories. Although insufficient data are available to classify monkeys, dogs and man appear to occupy the middle ground in which a small but significant parasympathetic component modulates airflow-induced bronchoconstriction. The fact that vagal activity can only partially account for airflow-induced bronchoconstriction in some asthmatic subjects suggests that vagal-dependent models may be of limited value in studying the human condition, but should prove valuable in elucidating the parasympathetic component of this mechanism. Although airflow-induced bronchoconstriction appears to be remarkably similar in guinea-pigs, dogs and humans, there are important differences concerning the potential role of specific mediators in producing airflow limitation. Concordant data from animal models and man suggest that: 1) airflow-induced bronchoconstriction is a basic mammalian response to airway desiccation; 2) airway drying stimulates and cooling inhibits this response; 3) hyperpnoea with dry air may damage the bronchial mucosa and contribute to this response; 4) biochemical mediators contribute to the development of this response; 5) vascular engorgement and airway oedema do not appear to be the primary effectors of this response, and in fact may antagonize it; 6) airway smooth muscle constriction is involved in the production of airflow-induced bronchoconstriction, and airway oedema may enhance its effect; and 7) airway and vascular responses to dehydration may protect against acute dry air-induced mucosal injury. Finally, although one must be cautious in extrapolating results from animals to humans, the similarities that do exist suggest that the investigation of airflow-induced bronchoconstriction in carefully selected animal models will continue to provide new insights concerning its development in humans.

The effect of bronchial blood flow on hyperpnea-induced airway obstruction and injury.

We examined the effect of bronchial blood flow (BBF) on hyperpnea-induced airway obstruction (HIAO) in dogs. HIAO in in situ isolated pulmonary lobes with or without BBF was monitored via a bronchoscope. An intravascular tracer in conjunction with morphometric analysis was used to document the efficacy of our occlusion technique. We found that (a) Occlusion of the bronchial artery abolished bronchovascular leakage, but did not alter HIAO; (b) HIAO occurred in postmortem dogs, and was attenuated by cooling; (c) absence of BBF did not cause mucosal damage, although hyperpnea-induced injury was enhanced in airways lacking BBF; (d) BBF did not affect either goblet/ ciliated cell ratios or hyperpnea-induced goblet cell degranulation; (e) ligation of the bronchial artery and hyperpnea each caused mast cell degranulation, and these effects were additive; (f) hyperpnea-induced leukocyte infiltration was reduced in the absence of BBF; and (g) ligation of the bronchial artery and hyperpnea with dry air each increased airway vessel diameter, and these effects were additive. We conclude that either impairment or absence of BBF abolishes bronchovascular leakage and increases hyperpnea-induced mucosal injury, but fails to affect HIAO. Based on these results we speculate that bronchovascular leakage protects the bronchial mucosa from excessive losses of heat and water, and inhibits mucosal damage.

The effects of alpha-adrenergic agonists on hyperpnea-induced airway obstruction in dogs.

Two alpha-adrenergic agonists that inhibit hyperpnea-induced airway obstruction (HIAO) in asthmatic subjects were used to examine the role of bronchial blood flow in the development of HIAO in canine periphery airways. A bronchoscope was used to record peripheral airway resistance (Rp) in anesthetized dogs before and after hyperpnea with dry air. Hyperpnea increased Rp 64 +/- 8% (mean +/- SE) above baseline. Treatment with norepinephrine (NOR) either before or at various times after hyperpnea inhibited HIAO (p < 0.01). We also found that NOR inhibited acetylcholine-induced bronchoconstriction. However, beta-adrenergic blockade with propranolol completely eliminated these effects. Thus, NOR inhibited HIAO in canine peripheral airways via the stimulation of beta-adrenergic receptors and the attenuation of airway smooth muscle contractility. In contrast, pretreatment with methoxamine (MX) decreased HIAO by approximately 25% when compared with the vehicle control, and this effect was completely eliminated by alpha-adrenergic blockade with phentolamine. Relative to NOR, MX provides weak protection against HIAO via the direct stimulation of alpha-adrenergic receptors and their subsequent effect on either mucus secretion or bronchovascular tone. We conclude that bronchial blood flow plays at best a minor role in the development of HIAO.