METHODS

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Studies to determine the prevalence and nature of postexercise airway inflammation were conducted at the conclusion of the 2001 and 2002 Iditarod, a 1,100-mile long race conducted each March stretching from Anchorage to Nome, Alaska. In the early portions of the race, ambient temperatures are typically between −10°C and 0°C. However, once over the Alaskan range and into the interior, temperatures can remain between −40°C and −10°C throughout the remainder of the race. In addition, many mushers prefer to run the dogs at night when the temperatures are lower, as this strategy reduces the risk of hyperthermia in the dogs. Thus, dogs will experience intermittent bouts of cold-air hyperpnea for 9 to 14 days while completing this race. For all studies, informed consent was obtained from the musher or authorized agent before the race and again at the conclusion of the race before initiating the preanesthetic fast. Dogs were allowed to rest with ad libitum food and water for a minimum of 12 hours after completing the race, followed by a 12-hour fast before bronchoscopy. Immediately before anesthetic induction, each dog underwent physical examination, including measurement of pulse and respiratory rates, rectal temperature, thoracic auscultation, palpation of the trachea and larynx, and coupage of the thorax (thoracic percussion using an open palm). No systemic illness or contraindications to anesthesia were found in any of the dogs examined. Dogs were anesthetized with an intravenous bolus of propofol (7 mg/kg; Abbott Laboratories, Abbott Park, IL) and were maintained with intermittent doses as needed to achieve suitable relaxation. An endotracheal tube was placed, and a 5-mm OD fiberoptic bronchoscope equipped with a video camera attached to the eyepiece was advanced into the lower airways. All large bronchi were individually inspected, and the presence and volume of mucus/exudates were scored using a scale of 0–3 (see Figure 1

Figure 1. Examples of bronchoscopic scoring grades: (A) bronchoscopy score = 0, no visible abnormalities; (B) bronchoscopy score = 1, mild accumulations of intraluminal material (note the crescent of mucus/exudates on the upper left); (C) bronchoscopy score = 2, moderate accumulations of intraluminal material throughout the airways; (D) bronchoscopy score = 3, extensive accumulation of intraluminal material throughout the airways.

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In the first study (conducted in 2001), 59 dogs from eight teams were examined 24 hours after completion of the race to estimate the prevalence of airway disease secondary to cold-weather strenuous exercise. In the second study (conducted in 2002), endoscopic and bronchoalveolar lavage findings in postexercise sled dogs (dogs examined 48 hours of completing the race, n = 10) were compared with a group of control sled dogs (dogs that had been trained throughout the winter but had not been exercised for at least 2 weeks at the time of examination, n = 12). After endoscopic examination and scoring (as described previously here), bronchoalveolar lavage fluid was obtained from a randomly selected sublobar airway from all dogs in this study. A sterile length of polyethylene tubing was advanced through the biopsy channel of the endoscope, and 20 ml of sterile Hank's phosphate-buffered saline was infused and immediately aspirated by hand. A total nucleated cell count and aerobic bacterial culture were performed within 24 hours of collection, and a differential cell count was performed on a cytocentrifuged slide preparation (200 μl, 1,000 rpm, 4 minutes) stained with a modified Wright-Giemsa stain. As our data were not normally distributed in all cases, endoscopic scores and differential nucleated cell concentrations were statistically compared using the Mann-Whitney Rank Sum test, with p < 0.05 considered significant. Subjective cytological evaluation was provided by an investigator (K.D.), who was blinded as to the physical examination, endoscopic findings, and sample differential cell concentrations.

RESULTS

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All dogs participating in this study had normal rectal temperature, pulse rate, and respiratory rate. Despite normal vital signs, thoracic coupage elicited coughs in 36% of the dogs. Thirty-six percent coughed during tracheal palpation, and 22% had abnormal lung sounds (crackles and/or wheezes) heard during thoracic auscultation. Of the dogs showing abnormal signs during physical examination, most had more than one abnormality. The mean endoscopic score was 1.41 ± 0.21 SEM. An abnormal accumulation of mucus or exudate was present in the lower airways of 48 out of 59 dogs (81%): 36% were classified as mild (endoscopic score 1), 32% were classified as moderate (endoscopic score 2), and 14% were considered severe (endoscopic score 3).

In the second study, the control dogs had a mean ± SEM endoscopic score of 0.67 ± 0.15, compared with a mean score of 1.8 ± 0.21 for the postexercise dogs (p = 0.0017). Postexercise dogs had greater bronchoalveolar lavage fluid (BALF)-nucleated cell concentrations (53.0 ± 7.6 × 10⁴ cells/μl versus 28.8 ± 8.0 × 10⁴ cells/μl in control dogs), with statistically significant increases in macrophages, lymphocytes, and eosinophils (Table 1)

DISCUSSION

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Human athletes who routinely experience hyperpnea in cold conditions have been reported to also have a high prevalence of peripheral airway inflammation and hyper-reactivity, leading some investigators to suggest that repeated penetration of incompletely conditioned air (air that has been incompletely warmed and humidified) into human peripheral airways can predispose these athletes to chronic airway disease similar to asthma (1–4, 8). In fact, this syndrome has been termed “ski asthma” in recognition of the similar phenotype and the most frequently studied human population affected by this syndrome. Studies using a canine laboratory model of peripheral airway exposure to incompletely conditioned air have reported the development of persistent airway obstruction, airway hyper-reactivity, impaired bronchodilation, airway inflammation (9, 10), and remodeling of the airway mucosa and lamina propria (11). In this study, we provide evidence that airway inflammation occurs under natural conditions in canine athletes. These observations provide additional support for the contention that repeated hyperpnea with cold air can injure peripheral airways and also identify a potential naturally occurring animal model of the analogous human condition.

During inhalation, heat and water vapor are transferred from the airway mucosa to the inspired air until the inspired air is warmed to body temperature and fully humidified. At rest and in temperate conditions, this process is completed in the upper airways so that there is minimal heat and water transfer that occurs in the intrapulmonary airways (12). However, studies in humans and horses have demonstrated that during hyperpnea, air that is not fully warmed (and, therefore, not completely humidified) penetrates into the intrapulmonary airways, particularly when the inspired air is cold (13, 14). As a result, heat and water is lost from the surface of the lower airways, resulting in airway mucosal cooling and probably desiccation (15, 16).

The complete pathogenesis of ski asthma is not known, but key features of this process have been suggested in human and laboratory animal experiments. Hyperosmolarity of the airway surface lining fluid has been demonstrated in dogs after peripheral airway exposure to cool, dry air (17) and has been suggested to also occur in humans during hyperpnea with cold, dry air (15, 16). Airway epithelial cells have been shown to release interleukin-8 (a key chemokine for neutrophils) in vitro in response to local hyperosmolarity (18), and mast cells have been shown to degranulate when exposed to hyperosmotic stimuli in vitro (19, 20). However, hyperosmolarity of the airway surface lining fluid may not be sufficient to produce inflammatory cell recruitment in vivo, as nebulization of hypertonic saline into canine peripheral airways failed to produce inflammatory cell influx (21). Thus, coincident airway cooling may be required for the development of ski asthma.

We do not believe that the airway pathology found in this study is the result of an infectious agent shared among the animals. All of the dogs were afebrile at the time of examination and as competing athletes were prohibited from receiving any medications that would have masked signs of illness. The dogs in the first study came from eight different racing teams that were separated by as many as 4 days, and the dogs in the second study came from three different teams, including one in which (comprising the entire control group) the dogs were geographically separated from each other. Thus, it is unlikely that the dogs had direct contact with each other before examination. Bacteria were observed in only one sample in the first study, and none of the samples from the second study yielded positive bacterial cultures. We cannot completely exclude the possibility of aeroallergens or other inhaled irritants as a factor in the respiratory disease in these dogs; however, in the case of aeroallergens, these particles are often observed in airway secretions that are examined microscopically. No foreign particles were noted in the samples obtained from these dogs. Thus, we believe that the airway disease identified in this population of racing sled dogs is a result of peripheral airway cooling and desiccation, resulting in airway injury and activation of local inflammatory pathways.

The accumulation of intraluminal material in the postrace dogs is likely due to a combination of both increased production of mucus and exudate, as well as decreased clearance of the intraluminal material. Both airway cooling and hyperosmolarity can provoke release of mucus by goblet cells and mucus glands (22). Goblet cells and mucous glands are found throughout the conducting airways in dogs (23), and thus, dogs are capable of considerable mucus production caused by airway cooling and desiccation, even in the peripheral airways. Airway cooling and desiccation also causes airway mucosal damage in humans (24), horses (25), and the canine laboratory model (26). Furthermore, repeated airway cooling and desiccation has been shown to produce extensive loss of ciliated mucosa, with the development of squamous metaplasia after only four daily challenges (11). These morphologic changes can be expected to impair mucociliary clearance, resulting in additional accumulation of intraluminal material. Although increased intraluminal mucus may help protect the airway from further desiccation, this theoretical benefit may be offset by the increase in airway resistance due to lumenal obstruction.

The absolute and differential nucleated cell concentrations in the BALF obtained from control dogs are comparable to those reported by our laboratory for untrained mongrel dogs (10), although the cytology of some of the control dogs suggested the presence of low-grade chronic inflammation. This inflammation may reflect previous training and/or racing injury that had not completely resolved in the 2 weeks of rest before examination. Increased BALF macrophage and eosinophil concentrations found in the postexercise dogs are in agreement with BALF obtained from dogs that received repeated airway cooling and desiccation (10). Furthermore, our results are in general agreement with similar studies in human winter athletes. Increased macrophage, lymphocyte, and eosinophil concentrations have been reported in endobronchial biopsies obtained from human winter athletes (27), and increased concentrations of BALF macrophages have also been reported in this population of humans (3). However, neutrophil concentrations were not significantly different from controls in this study, in contrast to previous studies using the canine laboratory model, which found increased neutrophil concentrations 24 hours after repeated peripheral airway challenge with inadequately conditioned air (10). It is important to note that in contrast to the laboratory studies, BALF in the second study was collected from the dogs in this study 48–50 hours after completion of the race. (The study was originally planned for examination of the dogs within 24 hours, but difficulties in moving personnel and equipment to Nome forced a delay.) We have previously shown that even after 4 days of severe laboratory challenge, resolution of airway neutrophilia takes less than 7 days (11). In the absence of continued airway challenge with inadequately conditioned air, the stimulus for release of neutrophil chemotactic factors undoubtedly waned and normal neutrophil turnover resulted in the loss of identifiable cells in the airway lumen. Thus, it is possible that the inadvertent delay in obtaining BALF from the postexercise dogs resulted in sufficient resolution of neutrophilia to prevent the detection of a statistically significant difference. This may also explain the lack of striking subjective abnormalities in the cytological evaluation of the samples.

Our demonstration of airway inflammation and intraluminal accumulation of mucus and exudate is in agreement with the hypothesis that some cases of ski asthma are the result of airway mucosal injury secondary to hyperpnea-induced airway hyperosmolarity. As recently summarized by Anderson and Holzer (28), excessive water loss by peripheral airways during hyperpnea can trigger a series of events leading to cough, mucus production, and airway obstruction. These signs (particularly cough) are commonly described after exercise in elite athletes regardless of whether they demonstrate airway hyper-reactivity consistent with exercise-induced asthma (29). Our findings thus add to the body of evidence suggesting that signs and symptoms consistent with exercise-induced asthma are phenomena resulting from exercise-induced airway injury and inflammation and can be provoked in otherwise healthy subjects by strenuous exercise.

Racing sled dogs are potentially useful animal models of ski asthma. Similar to cross-country skiers, sled dogs typically exercise below their maximum aerobic capacity for extended periods of time and in frigid conditions. In addition, typical sled dog teams are relatively uniform in their size, age, husbandry, environmental history, and genetic background. Thus, less intersubject variability would be expected compared with human athletes. Compared with humans, dogs can be subjected to more invasive techniques in the course of an investigation, and the canine peripheral airway responses to incompletely conditioned air measured in laboratory studies have remarkable fidelity to those reported in humans. Based on the results of these studies, these similarities extend to the naturally occurring disease that is shared by human and canine elite winter athletes.