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Equine veterinary journal2021; 53(6); 1188-1198; doi: 10.1111/evj.13403

Optimisation and validation of immunohistochemical axonal markers for morphological and functional characterisation of equine peripheral nerves.

Abstract: Horses are affected by various peripheral nerve disorders but defining their aetiology and pathophysiology is hampered by limited understanding of associated morphological and pathological changes and involvement of specific axonal types. Objective: To investigate the hypothesis that selected antibody markers, used in conjunction with various tissue processing methods, would enable identification of axons with different functional modalities within a range of equine peripheral nerves. Methods: Optimisation and validation study. Methods: A range of antibodies were evaluated immunohistochemically via fluorescence confocal microscopy in cadaver equine nerve samples of primary motor, mixed or primary sensory functions (recurrent laryngeal, phrenic and plantar digital) within formalin-fixed paraffin-embedded (FFPE) and formalin-fixed frozen (FFF) tissues subjected to different antigen retrieval protocols. Results: Immunohistochemistry of FFPE-derived nerve samples with selected antibodies and specific antigen retrieval methods enabled identification of myelinated and unmyelinated axons, cholinergic, sympathetic and peptidergic axons. The recurrent laryngeal and phrenic nerves are composed of myelinated cholinergic (motor), myelinated sensory fibres, unmyelinated adrenergic (sympathetic) axons and unmyelinated peptidergic (sensory) axons. In contrast, as expected, the plantar digital nerve had no myelinated motor fibres being mainly composed of myelinated sensory fibres, unmyelinated sympathetic and unmyelinated peptidergic sensory axons. Conclusions: Attempts specifically to label parasympathetic fibres were unsuccessful in any nerve examined in both FFPE and FFF tissues. Conclusions: A panel of antibody markers can be used to reveal morphological and functional properties of equine nerves. Future work should enable better characterisation of morphological changes in equine neuropathies at various stages of disease development.
© 2021 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.
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Publication Date: 2021-01-19 PubMed ID: 33338316DOI: 10.1111/evj.13403Google 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 research study focuses on improving the understanding of peripheral nerve disorders in horses, by experimenting with different antibody markers and tissue processing methods to identify specific axonal types linked to horse nerve disorders. Various antibodies were tested on nerve samples and categorized based on different functions to enable more detailed characterisation of equine neuropathies and the potential identification of disease development stages.

Research Objective and Hypothesis

  • The study intended to better comprehend disorders affecting the peripheral nerves in horses, which is currently limited due to a lesser understanding of linked morphological and pathological changes, as well as involved axonal types. The researchers hypothesized that using certain antibody markers and tissue processing techniques might aid in the identification of axons of different functional modalities within a range of equine peripheral nerves.

Methods and Experiments

  • This was an optimisation and validation study where different antibodies were evaluated using immunohistochemical methods and fluorescence confocal microscopy. The experiments were performed on cadaver equine nerve samples, which could be functioning primarily as motor, mixed, or sensory nerves.
  • The nerve samples were treated with formalin and then either embedded in paraffin (FFPE) or frozen (FFF), followed by different antigen retrieval protocols.

Results and Observations

  • By using specific antibodies and antigen retrieval methods on FFPE-treated nerve samples, the researchers were able to distinguish myelinated and unmyelinated axons, as well as cholinergic, sympathetic, and peptidergic axons.
  • The recurrent laryngeal and phrenic nerves mainly consisted of myelinated cholinergic (motor), myelinated sensory fibres, unmyelinated adrenergic (sympathetic) axons, and unmyelinated peptidergic (sensory) axons.
  • On the contrary, the plantar digital nerve, as anticipated, did not have myelinated motor fibres, and was primarily composed of myelinated sensory fibres, unmyelinated sympathetic, and unmyelinated peptidergic sensory axons.

Conclusions and Future Work

  • Attempts to specifically label parasympathetic fibres did not succeed with any of the nerve samples examined with both FFPE and FFF tissue treatments.
  • The study concludes by recommending the use of a panel of antibody markers to reveal the morphological and functional characteristics of equine nerves. The researchers suggest that future work could enable a more detailed understanding of morphological changes in equine neuropathies during the various stages of disease development.

Cite This Article

APA
Almuhanna AH, Cahalan SD, Lane A, Goodwin D, Perkins J, Piercy RJ. (2021). Optimisation and validation of immunohistochemical axonal markers for morphological and functional characterisation of equine peripheral nerves. Equine Vet J, 53(6), 1188-1198. https://doi.org/10.1111/evj.13403

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English
Volume: 53
Issue: 6
Pages: 1188-1198

Researcher Affiliations

Almuhanna, Abdulaziz H
  • Comparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Royal College Street, London, UK.
Cahalan, Stephen D
  • Comparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Royal College Street, London, UK.
Lane, Annette
  • Comparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Royal College Street, London, UK.
Goodwin, David
  • Comparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Royal College Street, London, UK.
Perkins, Justin
  • Comparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Royal College Street, London, UK.
Piercy, Richard J
  • Comparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Royal College Street, London, UK.

MeSH Terms

  • Animals
  • Axons
  • Horses
  • Immunohistochemistry
  • Nerve Fibers, Myelinated
  • Peripheral Nerves

Grant Funding

  • Horserace Betting Levy Board
  • Saudi Arabian Cultural Bureau (SACB)

References

This article includes 34 references
  1. Divers TJ, Mohammed HO, Cummings JF, Valentine BA, Delahunta A, Jackson CA. Equine Motor-Neuron Disease - Findings in 28 Horses and Proposal of a Pathophysiological Mechanism for the Disease.. Equine Vet J 1994;26:409-15.
  2. Duncan ID, Baker GJ. Experimental crush of the equine recurrent laryngeal nerve: a study of normal and aberrant reinnervation.. Am J Vet Res 1987;48:431-8.
  3. Aleman M, Williams DC, Brosnan RJ, Nieto JE, Pickles KJ, Berger J. Sensory nerve conduction and somatosensory evoked potentials of the trigeminal nerve in horses with idiopathic headshaking.. J Vet Intern Med 2013;27:1571-80.
  4. Cottrell DF, McGorum BC, Pearson GT. The neurology and enterology of equine grass sickness: a review of basic mechanisms.. Neurogastroenterol. Motil. 1999;11:79-92.
  5. Bilbao JM, Schmidt RE. Examination of the Peripheral Nerve Biopsy.. .
  6. Risk E, Shoja MM, Loukas M, Barbaro N, Spinner RJ. Nerve Fiber Types.. .
  7. Yamamoto Y, Atoji Y, Suzuki Y. Innervation of taste buds in the canine larynx as revealed by immunohistochemistry for the various neurochemical markers.. Tissue Cell 1997;29:339-46.
  8. Tsuda K, Shin T, Masuko S. Immunohistochemical Study of Intralaryngeal Ganglia in the Cat.. Otolaryng Head Neck 1992;106:42-6.
  9. Russo D, Bombardi C, Grandis A, Furness JB, Spadari A, Bernardini C. Sympathetic Innervation of the Ileocecal Junction in Horses.. Journal of Comparative Neurology 2010;518:4046-66.
  10. Corcoran BM, Mayhew IG, Hahn CN, Prince DR. Expression of vasoactive intestinal peptide, calcitonin gene-related peptide, substance P, and intermediate neurofilaments in nasal mucosal nerve fibers of horses without nasal disease.. Am J Vet Res 2000;61:1619-24.
  11. Russo D, Bombardi C, Castellani G, Chiocchetti R. Characterization of spinal ganglion neurons in horse (Equus caballus). A morphometric, neurochemical and tracing study.. Neuroscience 2011;176:53-71.
  12. Gay S, Jublanc E, Bonnieu A, Bacou F. Myostatin deficiency is associated with an increase in number of total axons and motor axons innervating mouse tibialis anterior muscle.. Muscle Nerve 2012;45:698-704.
  13. Corcoran BM, Jarvis S, Hahn CN, Mayhew IG. The distribution of nerve fibres immunoreactive for vasoactive intestinal peptide, calcitonin gene-related peptide, substance P and dopamine β-hydroxylase in the normal equine larynx.. Res Vet Sci 1999;67:251-9.
  14. Keast JJN. Visualization and immunohistochemical characterization of sympathetic and parasympathetic neurons in the male rat major pelvic ganglion.. Neurosci 1995;66:655-62.
  15. Robinson NJ. Consensus statements on equine recurrent laryngeal neuropathy: conclusions of the Havemeyer Workshop: September 2003, Stratford-upon-Avon, Warwickshire, UK.. Equine Vet Educ 2004;16:333-6.
  16. Anderson BH. Recurrent Laryngeal Neuropathy: Clinical Aspects and Endoscopic Diagnosis.. .
  17. Friede RL, Samorajski T. Axon caliber related to neurofilaments and microtubules in sciatic nerve fibers of rats and mice.. The Anatomical Record 1970;167:379-87.
  18. Quinlan TJ, Goulden BE, Barnes GR, Anderson LJ, Cahill JI. Innervation of the equine intrinsic laryngeal muscles.. N Z Vet J 1982;30:43-5.
  19. López-Plana C, Sautet JY, Pons J, Navarro G. Morphometric study of the recurrent laryngeal nerve in young ‘normal’ horses.. Res Vet Sci 1993;55:333-7.
  20. Duncan ID, Griffiths IR, Madrid RE. A light and electron microscopic study of the neuropathy of equine idiopathic laryngeal hemiplegia.. Neuropathol Appl Neurobiol 1978;4:483-501.
  21. Hahn CN, Matiasek K, Dixon PM, Molony V, Rodenacker K, Mayhew IG. Histological and ultrastructural evidence that recurrent laryngeal neuropathy is a bilateral mononeuropathy limited to recurrent laryngeal nerves.. Equine Vet J 2008;40:666-72.
  22. Duncan ID, Reifenrath P, Jackson KF, Clayton M. Preferential denervation of the adductor muscles of the equine larynx. II: Nerve pathology.. Equine Vet J 1991;23:99-103.
  23. Duncan ID, Griffths IR, McQueen A, Baker GO. The pathology of equine laryngeal hemiplegia.. Acta Neuropathol 1974;27:337-48.
  24. Duncan I, Griffiths I. Pathological changes in equine laryngeal muscles and nerves.. Proceedings of the 19th Annual Convention of the American Association of Equine Practitioners, Atlanta, Georgia 1973; 97-223.
  25. Cahill JI, Goulden BE. Equine laryngeal hemiplegia. Part III. A teased fibre study of peripheral nerves.. N Z Vet J 1986;34:181-5.
  26. Cahill JI, Goulden BE. Equine laryngeal hemiplegia. Part II. An electron microscopic study of peripheral nerves.. N Z Vet J 1986;34:170-5.
  27. Cahill JI, Goulden BE. Equine laryngeal hemiplegia. Part I. A light microscopic study of peripheral nerves.. N Z Vet J 1986;34:161-9.
  28. Draper ACE, Piercy RJ. Pathological classification of equine recurrent laryngeal neuropathy.. J Vet Intern Med 2018;32(4):1397-409.
  29. Duncan ID. The pathology of equine laryngeal hemiplegia.. PhD, University of Glasgow; 1975.
  30. Nair J, Streeter KA, Turner SMF, Sunshine MD, Bolser DC, Fox EJ. Anatomy and physiology of phrenic afferent neurons.. J Neurophysiol 2017;118:2975-90.
  31. Barcroft H, Bonnar WM, Edholm OG, Effron AS. On sympathetic vasoconstrictor tone in human skeletal muscle.. J Physiol-London 1943;102:21-31.
  32. Radovanovic D, Peikert K, Lindstrom M, Domellof FP. Sympathetic innervation of human muscle spindles.. J Anat 2015;226:542-8.
  33. Katz LM, Marr CM, Elliott J. Characterization of the responses of equine digital veins and arteries to calcitonin gene-related peptide.. Am J Vet Res 2011;72:975-81.
  34. Jones E, Vinuela-Fernandez I, Eager RA, Delaney A, Anderson H, Patel A. Neuropathic changes in equine laminitis pain.. Pain 2007;132:321-31.

Citations

This article has been cited 4 times.
  1. Amari M, Brioschi FA, Auletta L, Ravasio G. Ultrasound-Guided Radiofrequency Ablation and Pulsed Radiofrequency Treatment for Chronic Lameness Due to Distal Forelimb Disease in Horses: A Pilot Study. Animals (Basel) 2025 Aug 10;15(16).
    doi: 10.3390/ani15162341pubmed: 40867669google scholar: lookup
  2. Iżycka-Świeszewska E, Gulczyński J, Sejda A, Kitlińska J, Galli S, Rogowski W, Sigorski D. Remarks on Selected Morphological Aspects of Cancer Neuroscience: A Microscopic Photo Review. Biomedicines 2024 Oct 14;12(10).
    doi: 10.3390/biomedicines12102335pubmed: 39457647google scholar: lookup
  3. Amari M, Rabbogliatti V, Ravasio G, Auletta L, Brioschi FA, Riccaboni P, Dell'Aere S, Roccabianca P. Development of an ultrasound-guided radiofrequency ablation technique in the equine cadaveric distal limb: histological findings and potential for treating chronic lameness. Front Vet Sci 2024;11:1437989.
    doi: 10.3389/fvets.2024.1437989pubmed: 39247124google scholar: lookup
  4. Bordoni B, Escher AR, Duczyński M. Proposal for Manual Osteopathic Treatment of the Phrenic Nerve. Cureus 2024 Apr;16(4):e58012.
    doi: 10.7759/cureus.58012pubmed: 38606024google scholar: lookup
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