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.
Publication Date: 2025-12-15 PubMed ID: 41473106PubMed Central: PMC12745387DOI: 10.3389/fvets.2025.1673415Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
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
Researcher Affiliations
- 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.
- Division of Clinical Radiology, Department of Clinical Veterinary Science, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
- Divison of Veterinary Anatomy, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
- 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.
References
This article includes 49 references
- Pollitt CC. The illustrated horse’s foot: a comprehensive guide. Amsterdam: Elsevier Health Sciences; (2015).
- Elliott J, Bailey SR. A review of cellular and molecular mechanisms in endocrinopathic, sepsis-related and supporting limb equine laminitis. Equine Vet J. (2023) 55:350–75. doi: 10.1111/evj.13933,
- Orsini JA. Science-in-brief: equine laminitis research: milestones and goals. Equine Vet J. (2014) 46:529–33. doi: 10.1111/evj.12301,
- Moore RM, Eades SC, Stokes AM. Evidence for vascular and enzymatic events in the pathophysiology of acute laminitis: which pathway is responsible for initiation of this process in horses? Equine Vet J. (2004) 36:204–9. doi: 10.2746/0425164044877116,
- Morgan RA, Keen JA, Walker BR, Hadoke PWF. Vascular dysfunction in horses with endocrinopathic laminitis. PLoS One. (2016) 11:e0163815. doi: 10.1371/journal.pone.0163815,
- Shin T. Principles of magnetic resonance angiography techniques. Investig Magn Reson Imaging. (2021) 25:209–17. doi: 10.13104/imri.2021.25.4.209
- Belknap JK, Geor RJ. Equine laminitis. Hoboken, NJ, USA: John Wiley and Sons, Inc.: Wiley-Blackwell. (2017). 77–78.
- Griffin RL, McKnight AL, Rucker A, Bennett SD, Fiser DM. Initial experience using contrast magnetic resonance imaging in laminitic horses: 18 studies. J Equine Vet Sci. (2011) 31:685–92. doi: 10.1016/j.jevs.2011.05.013
- Underberg BA, Kaessmeyer S, Schweizer D, Drews B, Van der Vekens E. Non-contrast enhanced visualization of the equine foot vasculature in a cadaver model using time-of-flight sequence. Front Vet Sci. (2025) 12:1585940. doi: 10.3389/fvets.2025.1585940,
- Ramos-Vara JA, Miller MA. When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry-the red, brown, and blue technique. Vet Pathol. (2014) 51:42–87. doi: 10.1177/0300985813505879,
- Belknap JK, Geor RJ. Equine laminitis John Wiley and Sons, Inc. Hoboken, NJ, USA: Wiley-Blackwell; (2017). 455 p.
- Avdonin PP, Tsvetaeva NV, Goncharov NV, Rybakova EY, Trufanov SK, Tsitrina AA, et al. Von Willebrand factor in health and disease. Biochem (Mosc) Suppl A: Membr Cell Biol. (2021) 15:201–18. doi: 10.1134/S1990747821040036
- Patan-Zugaj B, Egerbacher M, Licka TF. Endotoxin-induced changes in expression of cyclooxygenase isoforms in the lamellar tissue of extracorporeally haemoperfused equine limbs. Anat Histol Embryol. (2020) 49:597–605. doi: 10.1111/ahe.12520,
- Weibel ER, Palade GE. New cytoplasmic components in arterial endothelia. J Cell Biol. (1964) 23:101–12. doi: 10.1083/jcb.23.1.101,
- Dong J, Moake JL, Nolasco L, Bernardo A, Arceneaux W, Shrimpton CN, et al. ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood. (2002) 100:4033–9. doi: 10.1182/blood-2002-05-1401
- Van Eps AW, Leise BS, Watts M, Pollitt CC, Belknap JK. Digital hypothermia inhibits early lamellar inflammatory signalling in the oligofructose laminitis model. Equine Vet J. (2012) 44:230–7. doi: 10.1111/j.2042-3306.2011.00416.x,
- Tsatsanis C, Androulidaki A, Venihaki M, Margioris AN. Signalling networks regulating cyclooxygenase-2. Int J Biochem Cell Biol. (2006) 38:1654–61. doi: 10.1016/j.biocel.2006.03.021,
- Schnabel CL, Wagner S, Wagner B, Durán MC, Babasyan S, Nolte I, et al. Evaluation of the reactivity of commercially available monoclonal antibodies with equine cytokines. Vet Immunol Immunopathol. (2013) 156:1–19. doi: 10.1016/j.vetimm.2013.09.012,
- Werner M, Chott A, Fabiano A, Battifora H. Effect of formalin tissue fixation and processing on immunohistochemistry. Am J Surg Pathol. (2000) 24:1016–9. doi: 10.1097/00000478-200007000-00014,
- Blaettler C, Kaessmeyer S, Grabherr S, Koch C, Schweizer D, Van der Vekens E. Post-mortem computed tomographic angiography in equine distal forelimbs: a feasibility study. Front Vet Sci. (2022) 9:868390. doi: 10.3389/fvets.2022.868390,
- Pollitt CC. Basement membrane pathology: a feature of acute equine laminitis. Equine Vet J. (1996) 28:38–46. doi: 10.1111/j.2042-3306.1996.tb01588.x,
- Fedchenko N, Reifenrath J. Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue - a review. Diagn Pathol. (2014) 9:221. doi: 10.1186/s13000-014-0221-9,
- Dietze K, Slosarek I, Fuhrmann-Selter T, Hopperdietzel C, Plendl J, Kaessmeyer S. Isolation of equine endothelial cells and life cell angiogenesis assay. Clin Hemorheol Microcirc. (2014) 58:127–46. doi: 10.3233/CH-141877,
- Ruiz FS, Alessi AC, Chagas CA, Pinto GA, Vassallo J. Immunohistochemistry in diagnostic veterinary pathology: a critical review. J Bras Patol Med Lab. (2005) 41:221–7. doi: 10.1590/s1676-24442005000400008
- Mebratie DY, Dagnaw GG. Review of immunohistochemistry techniques: applications, current status, and future perspectives. Semin Diagn Pathol. (2024) 41:154–60. doi: 10.1053/j.semdp.2024.05.001,
- Bussolati G, Leonardo E. Technical pitfalls potentially affecting diagnoses in immunohistochemistry. J Clin Pathol. (2008) 61:1184–92. doi: 10.1136/jcp.2007.047720,
- Grizzle WE, Stockard CR, Billings PE. The effects of tissue processing variables other than fixation on histochemical staining and immunohistochemical detection of antigens. J Histotechnol. (2001) 24:213–9. doi: 10.1179/his.2001.24.3.213
- Karikoski NP, McGowan CM, Singer ER, Asplin KE, Tulamo RM, Patterson-Kane JC. Pathology of natural cases of equine Endocrinopathic laminitis associated with hyperinsulinemia. Vet Pathol. (2015) 52:945–56. doi: 10.1177/0300985814549212,
- Peters SM. Frozen Section Technique. Frozen section technique – Part 4: Mohs and margin control. (2003). Available online at: https://www.pathologyinnovations.com/frozen-section-technique-4 (Accessed November 7, 2025).
- Giovannini E, Bonasoni MP, Bardaro M, Russello G, Carretto E, Zerbini A, et al. Postmortem histological freeze–thaw artifacts: a case report of a frozen infant and literature review. Forensic Sci Med Pathol. (2024) 20:1412–9. doi: 10.1007/s12024-023-00752-w,
- Taqi SA, Sami SA, Sami LB, Zaki SA. A review of artifacts in histopathology. J Oral Maxillofac Pathol. (2018) 22:279. doi: 10.4103/jomfp.JOMFP_125_15,
- Srinivasan M, Sedmak D, Jewell S. Review effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. (2002) 161:1961–71. doi: 10.1016/S0002-9440(10)64472-0
- Waguespack RW, Cochran AM, Belknap JK. Expression of the cyclooxygenase isoforms in the prodromal stage of black walnut-induced laminitis in horses. Am J Vet Res. (2004) 65:1724–9. doi: 10.2460/ajvr.2004.65.1724,
- Burns TA, Watts MR, Belknap JK, van Eps AW. Digital lamellar inflammatory signaling in an experimental model of equine preferential weight bearing. J Vet Intern Med. (2023) 37:681–8. doi: 10.1111/jvim.16662,
- Cassimeris L, Engiles JB, Galantino-Homer H. Interleukin-17A pathway target genes are upregulated in supporting limb laminitis. PLoS One. (2020) 15:e0232920. doi: 10.1371/journal.pone.0232920, n
- Cassimeris L, Armstrong C, Burger QC, Stokes S, van Eps A, Galantino-Homer H. Continuous digital hypothermia reduces expression of keratin 17 and 1L-17A inflammatory pathway mediators in equine laminitis induced by hyperinsulinemia. Vet Immunol Immunopathol. (2021) 241:110326. doi: 10.1016/j.vetimm.2021.110326,
- Leise BS, Faleiros RR, Watts M, Johnson PJ, Black SJ, Belknap JK. Hindlimb laminar inflammatory response is similar to that present in forelimbs after carbohydrate overload in horses. Equine Vet J. (2012) 44:633–9. doi: 10.1111/j.2042-3306.2011.00531.x,
- Loftus JP, Black SJ, Pettigrew A, Abrahamsen EJ, Belknap JK. Early laminar events involving endothelial activation in horses with black walnut-induced laminitis. Am J Vet Res. (2007) 68:1205–11. doi: 10.2460/ajvr.68.11.1205
- Blikslager AT, Yin C, Cochran AM, Wooten JG, Pettigrew A, Belknap JK. Cyclooxygenase expression in the early stages of equine laminitis: a cytologic study. J Vet Intern Med. (2006) 20:1191–6. doi: 10.1111/j.1939-1676.2006.tb00721.x,
- Dern K, van Eps A, Wittum T, Watts M, Pollitt C, Belknap J. Effect of continuous digital hypothermia on lamellar inflammatory Signaling when applied at a clinically-relevant timepoint in the oligofructose laminitis model. J Vet Intern Med. (2018) 32:450–8. doi: 10.1111/jvim.15027,
- Bishop-Bailey D, Pepper JR, Larkin SW, Mitchell JA. Differential induction of cyclooxygenase-2 in human arterial and venous smooth muscle role of endogenous prostanoids. Arterioscler Thromb Vasc Biol. (1998) 18:1655–61. doi: 10.1161/01.ATV.18.10.1655,
- Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol. (1998) 38:97–120. doi: 10.1146/annurev.pharmtox.38.1.97,
- Bernard MP, Bancos S, Sime PJ, Phipps RP. Targeting Cyclooxygenase-2 in Hematological malignancies: rationale and promise. Curr Pharm Des. (2008) 14:2051–60. doi: 10.2174/138161208785294654,
- Grabherr S, Djonov V, Friess A, Thali MJ, Ranner G, Vock P, et al. Postmortem angiography after vascular perfusion with diesel oil and a lipophilic contrast agent. Am J Roentgenol. (2006) 187:W515–23. doi: 10.2214/AJR.05.1394,
- Schwarzmaier SM, Knarr MRO, Hu S, Ertürk A, Hellal F, Plesnila N. Perfusion pressure determines vascular integrity and histomorphological quality following perfusion fixation of the brain. J Neurosci Methods. (2022) 372:109493. doi: 10.1016/j.jneumeth.2022.109493,
- Baldwin GI, Pollitt CC. Progression of venographic changes after experimentally induced laminitis. Vet Clin North Am Equine Pract. (2010) 26:135–40. doi: 10.1016/j.cveq.2009.12.005,
- D’Arpe L, Bernardini D. Digital venography in horses and its clinical application in Europe. Vet Clin North Am Equine Pract. (2010) 26:339–59. doi: 10.1016/j.cveq.2010.06.006
- Rucker A. Equine venography and its clinical application in North America. Vet Clin North Am Equine Pract. (2010) 26:167–77. doi: 10.1016/j.cveq.2009.12.008,
- Pollitt C.nEquine laminitis: current concepts. RIRDC Publication No 08/062 (2008). Available online at: https://agrifutures.com.au/product/equine-laminitis-current-concepts/ (Accessed July 25, 2025).
Citations
This article has been cited 0 times.Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
