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Veterinary ophthalmology2023; doi: 10.1111/vop.13107

The use of shotgun label-free quantitative proteomic mass spectrometry to evaluate the inflammatory response in aqueous humor from horses with uveitis compared to ophthalmologically healthy horses.

Abstract: The objective of this study was to use shotgun label-free tandem mass spectrometry (LF-MS/MS) to evaluate aqueous humor (AH) from horses with uveitis (UH) compared to ophthalmologically healthy horses (HH). Methods: Twelve horses diagnosed with uveitis based on ophthalmic examination and six ophthalmologically healthy horses (postmortem) purchased for teaching purposes. Methods: All horses received a complete ophthalmic examination and physical exam. Aqueous paracentesis was performed on all horses and AH total protein concentrations were measured with nanodrop (TPn) and refractometry (TPr). AH samples were analyzed with shotgun LF-MS/MS and proteomic data were compared between groups using Wilcoxon rank-sum test. Results: A total of 147 proteins were detected, 11 proteins had higher abundance in UH, and 38 proteins had lower abundance in UH. Proteins with higher abundance included apolipoprotein E, alpha-2-macroglobulin (A2M), alpha-2-HS-glycoprotein, prothrombin, fibrinogen, complement component 4 (C4), joining chain for IgA and IgM, afamin, and amine oxidase. There were positive correlations between TPn (p = .003) and TPr (p = .0001) compared to flare scores. Conclusions: Differential abundance of A2M, prothrombin, fibrinogen, and C4 indicate upregulation of the complement and coagulation cascade in equine uveitis. Proinflammatory cytokines and the complement cascade have potential as therapeutic targets for equine uveitis.
© 2023 The Authors. Veterinary Ophthalmology published by Wiley Periodicals LLC on behalf of American College of Veterinary Ophthalmologists.
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Publication Date: 2023-05-05 PubMed ID: 37144658DOI: 10.1111/vop.13107Google Scholar: Lookup
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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.

This study analyzes the protein differences in the eye fluid of horses suffering from eye inflammation, or uveitis, compared to healthy horses. Using a specific method of protein mass spectrometry, the researchers identified various proteins that were either more or less abundant in the fluid from affected horses, suggesting possible causes of inflammation and potential paths for treatment.

Study Purpose and Methods

  • This study sought to identify proteins in the aqueous humor, or eye fluid, using shotgun label-free tandem mass spectrometry. This technique is often used to detect and quantify proteins, and was used in this study to compare the protein content in the eye fluid of horses diagnosed with uveitis to that of healthy horses.
  • The study recruited twelve horses diagnosed with uveitis and six healthy horses, the latter being postmortem samples purchased for teaching purposes. The horses underwent a complete eye and physical examination. To obtain aqueous humor for analysis, a procedure known as paracentesis was performed on all horses. This involves using a needle to draw fluid from the eye.
  • The total protein concentrations in the collected aqueous humor samples were measured. The protein analysis was then performed using the mass spectrometry method, and the data obtained were compared between the two groups using a statistical method known as the Wilcoxon rank-sum test.

Study Results

  • The analysis detected a total of 147 proteins in the aqueous humor samples. Among these, 11 proteins were found to be more abundant in uveitis horses, and 38 proteins were less abundant as compared to the healthy horses.
  • The proteins showing higher frequency included apolipoprotein E, alpha-2-macroglobulin (A2M), alpha-2-HS-glycoprotein, prothrombin, fibrinogen, complement component 4 (C4), joining chain for IgA and IgM, afamin, and amine oxidase.
  • The results also revealed positive correlations between the total protein concentrations and flare scores, a clinical parameter used in uveitis evaluation, indicating that as the inflammation increased, so did the protein concentration.

Conclusion

  • The key findings noted differences in the amounts of A2M, prothrombin, fibrinogen, and C4, all proteins associated with inflammatory and clotting processes. As these were higher in the eye fluid of horses with uveitis, the results suggest that inflammation caused an upregulation or increase of these proteins, particularly the body’s complement and coagulation (clotting) response. By indicating a link with these proteins, the research points out the involvement of these systems in the development of uveitis in horses.
  • The researchers concluded that the findings provide potential directions for the treatment of equine uveitis by targeting proinflammatory proteins and the complement cascade, which is part of the immune system’s response to inflammation.

Cite This Article

APA
Terhaar HM, Henriksen ML, Mehaffy C, Hess A, McMullen RJ. (2023). The use of shotgun label-free quantitative proteomic mass spectrometry to evaluate the inflammatory response in aqueous humor from horses with uveitis compared to ophthalmologically healthy horses. Vet Ophthalmol. https://doi.org/10.1111/vop.13107

Publication

Veterinary ophthalmology
ISSN: 1463-5224
NlmUniqueID: 100887377
Country: England
Language: English

Researcher Affiliations

Terhaar, Hannah M
  • Comparative Ophthalmology, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
Henriksen, Michala de Linde
  • Comparative Ophthalmology, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
Mehaffy, Carolina
  • Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
Hess, Ann
  • Department of Statistics, College of Natural Sciences, Colorado State University, Fort Collins, Colorado, USA.
McMullen, Richard J
  • Equine Ophthalmology, Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, JT Vaughan Large Animal Teaching Hospital, Auburn, Alabama, USA.

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