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Frontiers in veterinary science2025; 12; 1673415; doi: 10.3389/fvets.2025.1673415

Cyclooxygenase-2 and von Willebrand factor-an immunohistochemical study of the equine foot with and without laminitis, post-mortem perfused with paraffin oil.

Abstract: Equine laminitis is a complex and potentially fatal disease characterized by severe vascular and inflammatory alterations within the equine foot. This study aimed to develop immunohistochemistry (IHC) protocols for the detection of cyclooxygenase-2 (COX-2) and von Willebrand factor (vWF) in equine feet with and without laminitis, post-mortem perfused with paraffin oil. Unassigned: A total of 12 equine forelimbs from 8 horses were utilized in this study, divided into two study cohorts: one with laminitis and the other as a non-laminitis control. To develop the IHC protocols thoroughly, the tissue samples were categorized into three groups: fresh, frozen-thawed, and frozen-thawed-perfused. IHC protocols for COX-2, a marker for inflammation, and vWF, a marker for vascular endothelial cells, were developed, respectively, optimized for use across these tissue groups. Samples from both study cohorts were processed and morphologically analyzed to assess tissue preservation and efficacy of immunostaining techniques. Unassigned: Once the optimal paraffin embedding as well as combination of blocking reagents, blocking duration and antibodies had been empirically determined, COX-2 and vWF immunostaining were successful in all tissue groups. Reducing the embedding time in paraffin from 24 h to 2 h and positioning the samples at a 45° angle within the embedding cassette optimized the cutting results for microtome sectioning. Immunostaining specificity was good and results of COX-2 and vWF in both study cohorts were comparable and reliable between all three tissue groups. COX-2 was considerably elevated in the laminits cohort, predominantly in basal and parabasal cells, fibroblasts, and endothelial cells. vWF immunostaining effectively highlighted the vascular endothelial cells, revealing vascular compression and dilation, especially in the laminitis cohort. Unassigned: This study provides evidence that COX-2 and vWF can be reliably detected in equine lamellar hoof tissues, after undergoing freezing, thawing, and perfusion treatments. The established IHC protocols represent valuable diagnostic tools for studying the inflammatory and vascular changes associated with the equine foot. These methods can be applied to post-mortem cadaver models that have been frozen and perfused with paraffin oil, thus reducing the number of live animals required for research.
Copyright © 2025 Underberg, Van der Vekens, Drews and Kaessmeyer.
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Publication Date: 2025-12-15 PubMed ID: 41473106PubMed Central: PMC12745387DOI: 10.3389/fvets.2025.1673415Google 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.

Overview

  • This study developed reliable immunohistochemistry (IHC) protocols to detect inflammation and vascular markers, cyclooxygenase-2 (COX-2) and von Willebrand factor (vWF), in horse feet tissue affected by laminitis and in healthy controls.
  • The protocols were optimized for post-mortem equine foot samples that were frozen, thawed, and perfused with paraffin oil, allowing detailed analysis of laminitis-associated changes without the use of live animals.

Background and Objective

  • Equine laminitis is a serious disease involving inflammation and vascular damage in the horse’s hoof, which can be fatal.
  • Understanding the disease mechanisms requires studying tissue markers of inflammation and vascular changes.
  • COX-2 is a known marker of inflammation, while vWF marks vascular endothelial cells.
  • The study aimed to develop and optimize IHC protocols to detect these markers in equine foot tissues that had undergone different preservation processes, facilitating analysis even in post-mortem samples.

Materials and Methods

  • Tissue Samples: 12 forelimbs from 8 horses were used, divided into two groups—laminitis affected and healthy controls.
  • Three tissue processing categories were used:
    • Fresh samples
    • Frozen-thawed samples
    • Frozen-thawed samples that were perfused with paraffin oil post-mortem
  • Development of IHC Protocols:
    • Protocols for COX-2 and vWF staining were optimized separately.
    • Blocking reagents, blocking times, and antibody concentrations were empirically tested to improve staining specificity.
    • Paraffin embedding was optimized by reducing embedding time from 24 hours to 2 hours and positioning tissue samples at a 45° angle for better microtome sectioning.

Results

  • Immunostaining for COX-2 and vWF was successful across all tissue groups (fresh, frozen-thawed, frozen-thawed-perfused).
  • COX-2 staining showed significantly higher expression in laminitis-affected tissue compared to controls, mainly in:
    • Basal and parabasal epidermal cells
    • Fibroblasts within the connective tissue
    • Endothelial cells lining blood vessels
  • vWF effectively highlighted vascular endothelial cells, revealing pathological vascular changes in laminitis samples such as compression and dilation of blood vessels.
  • The IHC results were consistent and comparable across all preservation groups, indicating the protocols are robust despite the freezing, thawing, and perfusion procedures.

Significance and Applications

  • This study confirms that COX-2 and vWF immunohistochemical detection is feasible and reliable in equine lamellar tissue that has undergone freezing, thawing, and paraffin oil perfusion.
  • The optimized IHC techniques allow researchers to use post-mortem frozen tissues, minimizing the need for live animal experimentation.
  • The methods enable detailed investigation of inflammatory and vascular mechanisms in laminitis, potentially improving understanding and diagnostic capabilities for this disease.
  • This approach facilitates the use of cadaver models for laminitis research, contributing to more ethical and practical study designs.

Cite This Article

APA
Underberg BA, Van der Vekens E, Drews B, Kaessmeyer S. (2025). Cyclooxygenase-2 and von Willebrand factor-an immunohistochemical study of the equine foot with and without laminitis, post-mortem perfused with paraffin oil. Front Vet Sci, 12, 1673415. https://doi.org/10.3389/fvets.2025.1673415

Publication

Frontiers in veterinary science
ISSN: 2297-1769
NlmUniqueID: 101666658
Country: Switzerland
Language: English
Volume: 12
Pages: 1673415
PII: 1673415

Researcher Affiliations

Underberg, Bianca A
  • Division of Clinical Radiology, Department of Clinical Veterinary Science, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
  • Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
Van der Vekens, Elke
  • Division of Clinical Radiology, Department of Clinical Veterinary Science, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Drews, Barbara
  • Divison of Veterinary Anatomy, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Kaessmeyer, Sabine
  • Divison of Veterinary Anatomy, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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