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Home / Equine Research Bank / Can J Vet Res / Evaluation of an in vitro degranulation challenge procedure ...
Canadian journal of veterinary research = Revue canadienne de recherche veterinaire1998; 62(2); 133-139;

Evaluation of an in vitro degranulation challenge procedure for equine pulmonary mast cells.

Abstract: Pulmonary mast cells (PMC) are important components of the inflammatory process in equine allergic lung diseases such as heaves. Very little, however, is known of the degranulation kinetics of these cells and thus, their pathophysiologic role remains largely speculative. The purpose of this study was to develop a repeatable protocol for in vitro equine PMC degranulation. Five mature horses (sex: 2 M, 3 F; age: 8.8 +/- 6.5 y), historically free of pulmonary disease and normal on clinical respiratory examination, arterial blood gas analysis, pulmonary mechanics testing and histamine inhalation challenge, were studied. Bronchoalveolar lavage was performed on 4 separate occasions, at least 2 d apart, in a different lung lobe on each occasion. The lavage fluid was concentrated by centrifugation. Cells were resuspended in modified HEPES/Tyrode, assessed for viability by Trypan blue exclusion, and PMC concentration determined. Cell inocula containing 30,000 PMC were incubated with 10(-8) to 6 x 10(-5) M A23187. Cells were then separated by centrifugation and histamine release (HR) was determined by fluorometric assay. The procedure was readily performed and yielded sufficient PMC for 30 to 60 inocula per lavage. Maximal HR (34.4 +/- 16.1%) was obtained with 10(-5) M A23187. The degranulation process was largely complete by 20 min but cell lysis was negligible. The challenge was repeatable within horse and produced a mean coefficient of variability of 23.0% following 20 min incubation with 10(-5) M A23187. We conclude that equine PMC degranulation can be repeatably performed in vitro and speculate that this protocol may be useful in further studies on the pathophysiology and treatment of equine allergic lung diseases.
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Publication Date: 1998-04-29 PubMed ID: 9553713PubMed Central: PMC1189460
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study focuses on developing a reliable method to stimulate the degranulation of pulmonary mast cells (PMC) in horses in a controlled or in vitro environment, in order to better understand the role of these mast cells in equine allergic lung diseases.

Understanding the Research

  • The core part of the research revolves around the degranulation of pulmonary mast cells (PMC). These cells are integral to inflammation in horse lung diseases, including heaves, an allergic respiratory disease. Despite being key players in these diseases, there is limited understanding of their degranulation behaviour, and thus their precise pathophysiologic role remains largely speculative.
  • This study aims to provide some clarity by developing a repeatable in vitro (or laboratory-controlled) method for inciting and studying PMC degranulation.

Study Methodology

  • Researchers used five mature horses, all of which had no history of pulmonary disease. They underwent bronchoalveolar lavage—a procedure for sampling cells from the lung—on four separate occasions.
  • The cells harvested from this lung lavage were then concentrated by centrifugation and resuspended in a specialized solution for assessment of vitality through a procedure known as Trypan blue exclusion.
  • These cells were then stimulated, or ‘challenged’, with a compound named “A23187” in increasing concentrations. This substance induces the cells to degranulate or release inflammatory compounds. Post-challenge, the cells were separated by another round of centrifugation and the amount of histamine released, indicative of degranulation degree, was measured using a fluorometric assay.

Results and Conclusion

  • The study produced successful and repeatable results with the formulation of the in vitro degranulation challenge. It provided a sufficient quantity of PMC for multiple challenges.
  • Significant histamine release was achieved with a specific concentration of A23187, suggesting that the protocol was effective at inducing PMC degranulation. The process was largely complete within 20 minutes, with minimal cell lysis, thus maintaining the cells intact for further analysis.
  • Ultimately, the researchers conclude that in-vitro degranulation of equine PMCs is possible and repeatable. They suggest that this newfound technique will be helpful in future studies exploring the pathophysiology and potential treatments for equine allergic lung diseases.

Cite This Article

APA
Hare JE, Viel L, Conlon PD, Marshall JS. (1998). Evaluation of an in vitro degranulation challenge procedure for equine pulmonary mast cells. Can J Vet Res, 62(2), 133-139.

Publication

Canadian journal of veterinary research = Revue canadienne de recherche veterinaire
ISSN: 0830-9000
NlmUniqueID: 8607793
Country: Canada
Language: English
Volume: 62
Issue: 2
Pages: 133-139

Researcher Affiliations

Hare, J E
  • Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph.
Viel, L
    Conlon, P D
      Marshall, J S

        MeSH Terms

        • Administration, Inhalation
        • Animals
        • Bronchoalveolar Lavage Fluid / cytology
        • Calcimycin / pharmacology
        • Carbon Dioxide / blood
        • Cytoplasmic Granules / physiology
        • Female
        • Histamine / administration & dosage
        • Histamine / pharmacology
        • Histamine Release
        • Horses
        • In Vitro Techniques
        • Lung / physiology
        • Male
        • Mast Cells / drug effects
        • Mast Cells / physiology
        • Mast Cells / ultrastructure
        • Oxygen / blood
        • Partial Pressure
        • Respiration / drug effects
        • Respiration / physiology
        • Respiratory Function Tests / veterinary

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