Source: OKLAHOMA STATE UNIVERSITY submitted to
EFFECTS OF COLD WEATHER EXERCISE ON EQUINE LUNG FUNCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0196006
Grant No.
(N/A)
Project No.
OKL02512
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2003
Project End Date
Sep 30, 2009
Grant Year
(N/A)
Project Director
Davis, M. S.
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
VETERINARY MEDICINE
Non Technical Summary
(N/A)
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30538101020100%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1020 - Physiology;
Goals / Objectives
This project consists of two broad objectives. This first objective is to determine the deleterious effects of strenous cold weather exercise on airway function in horses. This will include a comprehensive evaluation of the upper and lower airways, including the effects on pulmonary mechanics, mucosal barrier function, clearance of inhaled debris, and resistance to airborne infectious agents. The second objective will be to identify husbandry, management, and pharmacological interventions that will prevent serious deleterious effects of strenuous cold weather exercise on equine airways.
Project Methods
All studies will use horses trained to perform controlled, strenuous exercise on a high-speed treadmill. In all cases, the experimental designs are expected to consist of a simple randomized crossover design, in which horses perform brief periods of strenuous exercise while breathing either warm or cold air. In the latter case, cold air will be generated using a large capacity air chiller that delivers sufficient cold air to the horse through a loose-fitting facemask. Various endpoints will be evaluated as appropriate. Airway mechanical properties will be measured using impulse oscillometry to provide comprehensive quantification of the work of breathing. Barrier function and mucociliary clearance will be evaluated by measuring the clearance of radioaerosols, histology, and ultramorphometry. Local immunology will be evaluated through the quantification of local cytokine production, cellular responses, and measurement of other key mediators. The specific endpoints pertaining to local immunology are expected to evolve as the overall availability of equine-specific reatents and protocols improves. The second set of studies will be tailored to the important findings in the first set. When specific biochemical pathways are identified, pharmaceuticals that are known or believed to interrupt those pathways will be tested for efficacy in blocking the most important deleterious effects. The specific relationship betwee exercise severity, ambient temperature, and airway damange will be quantified in order to determine a continuous scale for permitted vs inadvisable exercise under specific climatic conditions.

Progress 10/01/03 to 09/30/09

Outputs
OUTPUTS: The major outcome of this project has been the development of a physiological non-exercising model of cold air lung injury. Initial attempts to use carbon dioxide rebreathing to stimulate hyperpnea were surprisingly unsuccessful. The rebreathing system was constructed using the existing cold air generation system converted to a closed circuit and equipped with a sealed mask and exogenous carbon dioxide source to prime the system. Inspired CO2 was monitored continuously at the mask. Using this system, horses demonstrated only minimal hyperpnea response, despite inspired CO2 concentrations as high as 80 mm Hg. Although blood gas measurements were not performed concurrently, we suspect that the horses were able to buffer the CO2 sufficiently to prevent hyperpnea. A second attempt to develop a non-exercise model employed continuous intravenous infusion of lobeline (a chemical stimulant of the respiratory center in mammals) to create a controlled 5 min period of hyperpnea similar to the period of hyperpnea in our exercise model. This procedure was well-tolerated by the horses, allowing us to perform a crossover study using room temperature or subfreezing inspired air, followed by bronchoalveolar lavage 24 hr after challenge. This model failed to demonstrate the cellular influx or alterations in cytokine expression observed in the exercise model. The third, and ultimately successful, method for developing a non-exercising model of cold air lung injury was the use of hypertonic aerosols to simulate the water loss from the airway surface that also occurs during hyperpnea with cold air. We used 2 compounds (saline and mannitol) at 4 different concentrations (300, 375, 450, and 525 mosm) to determine the appropriate challenge. Saline was used because it is the most physiological approach, since it would increase the airway surface fluid (ASF) osmolarity using additional amounts of osmoles normally present in the ASF. The lowest concentration is iso-osmolar with ASF, and served as a control, whereas the highest concentration is the highest concentration achieved during experimental cold air hyperpnea. Mannitol was used because during cold air hyperpnea, the airway surface cools as well as becomes hyperosmolar, and it is believed that this cooling slows the cellular processes that help reestablish normal osmolarity, thus prolonging the hyperosmolar stimulus. By using mannitol instead of saline to create the hyperosmolarity, the re-establishment of normal osmolarity is delayed. This strategy proved to be appropriate, as we saw little response to any concentration of saline aerosol. However, the mannitol aerosol demonstrated a distinct dose-response relationship in acute bronchoconstriction. The highest concentration of mannitol also produced a doubling of the concentration of neutrophils in the airways, as well as upregulation of interleukin-8. All of these phenomena have been demonstrated in the analogous studies of exercise while breathing cold air, confirming that aerosolized hypertonic mannitol is a useful non-exercising substitute model of cold air-induced airway inflammation. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Target audiences are human and veterinary researchers in the field of airway disease and exercise physiology. PROJECT MODIFICATIONS: Methodology was altered as described above to meet the study objectives.

Impacts
The primary outcome of the recent activity in this project is the decision by the investigator to combine the hypertonic aerosol model with the exercising model to advance the study of cold air-induced airway injury in horses. Validation of a non-exercising model of cold air-induced airway injury was an economic necessity to permit the study of the effects of cold air on airway resistance to infection. We had previously proposed the rebreathing model in grants submitted to NIH and DoD. These grants will be resubmitted using the hypertonic aerosol model as our intermediate model of cold air-induced airway injury. Our results, while partially fulfilling this requirement, demonstrate that although some mechanistic work can be conducted using the hypertonic aerosol model, the exercising model will remain necessary to confirm the findings. The results of this study provide further evidence that strenuous exercise in cold climates can induce non-septic airway inflammation characteristic of inflammatory airway disease (IAD) in horses. Reinforcing this information will underscore the importance of environment on respiratory health in horses, and provide the foundation for the development of preventative and management tools to minimize respiratory disease in athletic horses.

Publications

  • No publications reported this period


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Studies were conducted to evaluate the cytokine expression in the non-exercising model of exercise-induced airway injury, using aerosolized hypertonic mannitol. This model, originally developed during the previous year of this project, is expected to reproduce the relevant immunological milieu that has been previously documented in airways of horses that exercise while breathing cold air. Development of a non-exercising model will facilitate a more economical approach to investigating potential consequences of these phenomena. These data have been presented to colleagues in the form of invited talks pertaining to airway health and disease in humans and domestic animals. PARTICIPANTS: Sabrina Cummings, DVM (PhD student, for whom this work will form part of her dissertation) Heather Denham (DVM student participating in NIH-sponsored summer research program) Brittany Fanning (Undergraduate pre-veterinary student worker) Danielle Page (Undergraduate pre-veterinary student worker) Patricia O'Neill (Undergraduate pre-veterinary student worker) TARGET AUDIENCES: Target audiences will be veterinary and human researchers in the field of airway disease and exercise physiology. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Exposures must be conducted during the summer in order to minimize the background effects of routine breathing of cold air. Unfortunately, coincident with the scheduled challenges, our laboratory facilities were closed for 8 weeks due to asbestos contamination. Exposures were conducted during the late summer, and sample analysis for cytokine expression via qPCR is on-going.

Publications

  • No publications reported this period


Progress 10/01/06 to 09/30/07

Outputs
Progress on USDA OKL02512 (Effects of Cold Weather Exercise on Equine Lung Function, PI: Davis, M.S.) is in the final stages of completion, with remaining samples currently being analyzed for cytokine mRNA expression. As reported in the previous progress report, Objective #1 was completed on schedule. The second objective for Year 1 has also been completed, and found that breathing cold air during strenuous exercise caused a modest increase in non-stimulated oxidative burst activity of airway leukocytes, but no detectable difference in the basal or stimulated phagocytosis activity. These results were presented in abstract form at the American College of Sports Medicine annual scientific meeting in May, 2007 The objective for year 2 (test the efficacy of mast cell stabilizing drugs to block hypertonicity-induced airway responses) was postponed in favor of an attempt to develop a more physiological non-exercising model of cold air lung injury using chemically-induced hyperpnea. Initial attempts to use carbon dioxide rebreathing to stimulate hyperpnea were surprisingly unsuccessful. The rebreathing system was constructed using the existing cold air generation system converted to a closed circuit and equipped with a sealed mask and exogenous carbon dioxide source to prime the system. Inspired CO2 was monitored continuously at the mask. Using this system, horses demonstrated only minimal hyperpnea response, despite inspired CO2 concentrations as high as 80 mm Hg. Although blood gas measurements were not performed concurrently, we suspect that the horses were able to buffer the CO2 sufficiently to prevent hyperpnea. Higher concentrations of inspired CO2 were not used, as we concluded that such a model would not be accepted by reviewers due to the magnitude of local airway acidosis that was undoubtedly present. Instead, we employed continuous intravenous infusion of lobeline (a chemical stimulant of the respiratory center in mammals) to create a controlled 5 min period of hyperpnea similar to the period of hyperpnea in our exercise model. This procedure was well-tolerated by the horses, allowing us to perform a crossover study using room temperature or subfreezing inspired air, followed by bronchoalveolar lavage 24 hr after challenge. This model failed to demonstrate the cellular influx or alterations in cytokine expression observed in the exercise model. These results suggest that hyperpnea alone is insufficient to trigger the cellular events of cold air-induced lung injury, possibly due to the availability of cardiac output to be redirected to the airways to enhance thermal equilibration. (During exercise, cardiac output is largely redirected to working muscle, at the expense of other tissues.)

Impacts
The primary outcome of the recent activity in this project is the decision by the investigator to combine the hypertonic aerosol model with the exercising model to advance the study of cold air-induced airway injury in horses. As stated in our previous progress report, validation of a non-exercising model of cold air-induced airway injury was an economic necessity to permit the study of the effects of cold air on airway resistance to infection. We had previously proposed the rebreathing model in grants submitted to NIH and DoD. These grants will be resubmitted using the hypertonic aerosol model as our intermediate model of cold air-induced airway injury. Our results, while partially fulfilling this requirement, demonstrate that although some mechanistic work can be conducted using the hypertonic aerosol model, the exercising model will remain necessary to confirm the findings. The results of this study provide further evidence that strenuous exercise in cold climates can induce non-septic airway inflammation characteristic of inflammatory airway disease (IAD) in horses. Reinforcing this information will underscore the importance of environment on respiratory health in horses, and provide the foundation for the development of preventative and management tools to minimize respiratory disease in athletic horses.

Publications

  • No publications reported this period


Progress 10/01/05 to 09/30/06

Outputs
Progress on USDA OKL02512 (Effects of Cold Weather Exercise on Equine Lung Function, PI: Davis, M.S.) is slightly behind schedule, with completion of the first objective, but not the second objective scheduled for Year 1. The first objective (Reproduce the phenomenon of cold air-induced airway immunosuppression using aerosolized hypertonic solutions) was completed with surprising results. Hypertonic mannitol aerosol produced an acute dose-dependent increased in pulmonary resistance, similar to the responses reported in other species and confirming the bronchoconstrictive nature of hypertonic mannitol. However, equimolar aerosols of sodium chloride did NOT produce acute airway obstruction, suggesting that hypertonicity alone is insufficient to provoke bronchoconstriction in horses. We believe that equine airways are capable of rapidly equilibrating ion concentrations, and thus the hypertonicity induced by the hypertonic saline aerosol was transient compared to that induced by mannitol, which is not readily transported across epithelial membranes. Neither type of hypertonic aerosol induced mucosal damage and late phase bronchoconstriction as is reported with exercise while breathing cold air, illustrating both the limited capacity for hypertonic aerosols to simulate exercise-induced airway damage but also demonstrating that the pathophysiology of the latter is mediated by more than transient airway hypertonicity. The second Objective scheduled for Year 1 (Determine the functional consequences of cold air-induced airway immunosuppression on alveolar macrophage responses to pathogens) has not been completed. Oxidative burst measurements have been completed, and demonstrate a limited pro-inflammatory response to cold air. Phagocytosis assays have been completed, but complete data analysis is on-going and is expected to be completed by the end of the calendar year.

Impacts
The results of this study provide further evidence that strenuous exercise in cold climates can induce non-septic airway inflammation characteristic of inflammatory airway disease (IAD) in horses. Reinforcing this information will underscore the importance of environment on respiratory health in horses, and provide the foundation for the development of preventative and management tools to minimize respiratory disease in athletic horses.

Publications

  • No publications reported this period


Progress 10/01/04 to 09/30/05

Outputs
Progress on USDA OKL02512 (Oxidative Stress in Racehorses, PI: Davis, M.S.) has proceeded according to schedule. All data collection and analysis has been completed. Both ethane and pentane (lipid peroxidation products of omega-3 and omega-6 fatty acids, respectively) could be detected in the breath horses, but in resting horses the coefficients of variation were quite high (> 100% for ethane, 35% for pentane). During gas chromatography, a discrete ethane peak was often obscured by a very large methane peak. Thus, pentane is the more useful oxidative biomarker in exhaled breath in horses. Glutathione redox ratio was measured, and demonstrated good reproducibility (CV<20%). Pentane and GRR had acceptable correlation throughout training, but the correlation was lost in measurements taken immediately after exercise. This is likely due to the unstable nature of redox homeostasis immediately after exercise and the differing half-lives of the markers. These features should be considered when interpreting these markers in future studies. Our data demonstrated moderate increases in oxidative stress upon initiation of training, but a return to pre-training baseline at peak fitness. These observations are in agreement with in vitro and in vivo work demonstrating the induction of antioxidant systems in response to oxidative stress. Our data demonstrates that although aerobic training can initially induce oxidative stress, healthy subjects are capable of adapting to these demands and specific antioxidant supplementation is not necessary. Acetone production increased throughout training and in response to exercise. Acetone is the spontaneous decarboxylation product of acetoacetic acid, which is in turn in equilibrium with beta-hydroxybutyrate in the bloodstream. Thus, release of acetone in exhaled breath can increase either through an increase in total ketones or a decrease in the redox status of the mitochondria. Total ketones and/or the fractions of the various ketone bodies in blood were not measured, so we can not conclude which of these changes was present. The increase in acetone post-exercise was likely due to post-exercise ketosis, a phenomenon widely described in other species. However, we have no explanation for the training-induced effect on acetone. We were able to reliably detect ethanol and methanol in exhaled breath. These alcohols are produced by intestinal fermentation, and though they are not biologically important per se, they may have critical importance in management of competitive equine athletes. In recent years, horse racing-related agencies have issued specific calls for projects to address detection of illegal administration of alcohol. Our results demonstrate that ENDOGENOUS production of alcohols is not only possible, but increased under conditions similar to those planned by regulatory officials (including a 4-fold increase in ethanol release immediately after exercise). Thus, the quantity and pattern of endogenous alcohol production must be examined before legally binding testing can be instituted.

Impacts
The results of this study illustrate the potential utility of non-invasive breath testing in horses for the evaluation of a number of physiological processes, including oxidative stress, metabolism, and forensic testing. The most immediate impact will be in the field of forensic testing, in which breath quantitation of ethanol will be the foundation of testing equine athletes during regulated competition.

Publications

  • No publications reported this period


Progress 10/01/03 to 09/30/04

Outputs
Progress on USDA OKL02512 (Effects of Cold Weather Exercise on Equine Lung Function, PI: Davis, M.S.) has proceeded according to schedule. During the fall of 2004, experiments were conducted to examine the effects of training and exercise on physiological and oxidative stress in horses. Sample for analysis of glutathione redox ratio, soluble antioxidants, cortisol, and breath-based biomarkers were collected before training, during training, and immediately before, after, and 5 hr after strenuous exercise. Some sample analysis has occurred, and other samples await analysis. Thus far, there is little evidence that training or exercise increases physiological or oxidative stress, in contrast to reports of such stresses in other studies. These results suggest that management factors may be important in the development of adverse responses to exercise, rather than the exercise itself. These conclusions are preliminary, as analysis of samples from the research horses is incomplete. Furthermore, these same analyses will be repeated in active, privately-owned equine athletes to verify the findings and eliminate insensitivity of our laboratory techniques as a factor in our findings.

Impacts
The results of these studies will determine whether racehorses suffer from oxidative stress, and if so, what aspect of their management is most respoinsible for this stress. Our conclusions will enable horsemen to make rational, informed decisions regarding the cost-benefit relationship of their management practices, including the possibility of antioxidant supplementation during training or racing.

Publications

  • No publications reported this period


Progress 10/01/02 to 09/30/03

Outputs
Began in October. No progress to report this period.

Impacts
New in October. None to report this period.

Publications

  • No publications reported this period