FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / PLoS One / Development of a constant pressure perfused ex vivo model of...
PloS one2021; 16(5); e0251530; doi: 10.1371/journal.pone.0251530

Development of a constant pressure perfused ex vivo model of the equine larynx.

Abstract: Distal axonopathy is seen in a broad range of species including equine patients. In horses, this degenerative disorder of the recurrent laryngeal nerve is described as recurrent laryngeal neuropathy (RLN). The dysfunctional innervation of the cricoarytenoideus dorsalis muscle (CAD) leads to a loss of performance in affected horses. In general, ex vivo models of the larynx are rare and for equine patients, just one short report is available. To allow for testing new therapy approaches in an isolated organ model, we examined equine larynges in a constant pressure perfused setup. In order to check the vitality and functionality of the isolated larynx, the vessels´ reaction to norepinephrine (NE) and sodium nitroprusside (NP) as vasoactive agents was tested. Additionally, the contractility of the CAD was checked via electrical stimulation. To determine the extent of hypoxic alterations, lactate dehydrogenase (LDH) and lactate were measured and an immunofluorescent analysis of hypoxia-inducible factor (HIF-1α), a key transcription factor in hypoxia, was performed. For this, a hypoxia-induced cell culture for HIF-1α was developed. The application of NE led to an expected vasoconstriction while NP caused the expected vasodilation. During a perfusion period of 352 ±20.78 min, LDH values were in the reference range and lactate values slightly exceeded the reference range at the end of the perfusion. HIF-1α nuclear translocation could reliably be detected in the hypoxia-induced cell cultures, but not in sections of the perfused CAD. With the approach presented here, a solid basis for perfusing equine larynges was established and may serve as a tool for further investigations of equine larynx disorders as well as a transferrable model for other species.
Get Full Text
Publication Date: 2021-05-20 PubMed ID: 34014952PubMed Central: PMC8136745DOI: 10.1371/journal.pone.0251530Google 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.
LinkedInCopy
  • 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 research article details the development and testing of a constantly pressurized ex vivo model of a horse’s larynx, useful for studying and testing treatments for recurrent laryngeal neuropathy in horses.

Study Focus

  • The researchers aimed to create an ex vivo model of the equine larynx that would facilitate studies related to recurrent laryngeal neuropathy (RLN).
  • Recurrent laryngeal neuropathy is a degenerative nerve disorder seen in horses, which seriously affects their performance. It is specifically related to dysfunction of the cricoarytenoideus dorsalis muscle (CAD).

Ex Vivo Model Development

  • The team developed a model under constant pressure perfusing conditions. This setup emulated the physiological conditions of a horse’s larynx.
  • A key aspect of the model testing was ensuring that the isolated larynx remained vital and functional. The team used norepinephrine (NE) and sodium nitroprusside (NP) as vasoactive agents to test the vessel’s reactions.
  • The scientists also examined the contractility of the CAD via electrical stimulation.

Measurement and Testing of Hypoxic Conditions

  • To assess hypoxic alterations, certain parameters were measured such as lactate dehydrogenase (LDH) and lactate—both typically associated with such conditions.
  • A part of the study involved developing a hypoxia-induced cell culture for hypoxia-inducible factor (HIF-1α), a key factor in hypoxic conditions.
  • LDH values remained within the reference range, and lactate values slightly surpassed the reference range towards the end of the perfusion period. This suggests minimal cell damage.

Results and Future Applications

  • The application of NE led to vasoconstriction, and NP induced vasodilation, both congruent with expected outcomes.
  • HIF-1α nuclear translocation could be consistently observed in hypoxia-induced cell cultures, but not in samples of the perfused CAD. This may indicate a lack of severe hypoxic conditions in the perfused tissues.
  • The developed ex vivo model can now provide a basis for future studies into equine larynx disorders and could be an adaptable model for studying similar disorders in other species.

Cite This Article

APA
Otto S, Michler JK, Dhein S, Mülling CKW. (2021). Development of a constant pressure perfused ex vivo model of the equine larynx. PLoS One, 16(5), e0251530. https://doi.org/10.1371/journal.pone.0251530

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 16
Issue: 5
Pages: e0251530
PII: e0251530

Researcher Affiliations

Otto, Sven
  • Institute of Veterinary Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, Leipzig University, Germany.
Michler, Jule K
  • Institute of Veterinary Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, Leipzig University, Germany.
Dhein, Stefan
  • Fachdienst Gesundheit, Altenburg, Germany.
Mülling, Christoph K W
  • Institute of Veterinary Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, Leipzig University, Germany.

MeSH Terms

  • Animals
  • Cells, Cultured
  • Horse Diseases / pathology
  • Horses / physiology
  • Hypoxia / pathology
  • Hypoxia / veterinary
  • Hypoxia-Inducible Factor 1, alpha Subunit / analysis
  • Laryngeal Diseases / pathology
  • Laryngeal Diseases / veterinary
  • Laryngeal Muscles / pathology
  • Laryngeal Nerves / pathology
  • Larynx / pathology
  • Perfusion

Conflict of Interest Statement

The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: SO received a stipend through Med-El Company, Austria. There are no patents, products in development or marketing products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.The pilot study of this project was presented at the BMT (Biomedizinische Technik) congress “Dreiländertagung der Deutschen, Schweizerischen und Österreichischen Gesellschaft für Biomedizinische Technik, Graz, Austria” and published as a case study in the congress proceedings [42].

References

This article includes 42 references
  1. Draper ACE, Piercy RJ. Pathological classification of equine recurrent laryngeal neuropathy.. J Vet Intern Med 2018 Jul;32(4):1397-1409.
    doi: 10.1111/jvim.15142pmc: PMC6060325pubmed: 29691904google scholar: lookup
  2. Dixon PM, Hahn CN, Barakzai SZ. Recurrent laryngeal neuropathy (RLN) research: where are we and to where are we heading?. Equine Vet J 2009 Apr;41(4):324-7.
    doi: 10.2746/042516409x423082pubmed: 19562891google scholar: lookup
  3. Ducharme NG, Cheetham J, Sanders I, Hermanson JW, Hackett RP, Soderholm LV, Mitchell LM. Considerations for pacing of the cricoarytenoid dorsalis muscle by neuroprosthesis in horses.. Equine Vet J 2010 Sep;42(6):534-40.
    doi: 10.1111/j.2042-3306.2010.00115.xpubmed: 20716194google scholar: lookup
  4. Lechartier A, Rossignol F, Brandenberger O, Vitte A, Mespoulhès-Rivière C, Rossignol A, Boening KJ. Mechanical comparison of 3 anchoring techniques in the muscular process for laryngoplasty in the equine larynx.. Vet Surg 2015 Apr;44(3):333-40.
    doi: 10.1111/j.1532-950X.2014.12248.xpubmed: 25069790google scholar: lookup
  5. Rossignol F, Brandenberger O, Perkins JD, Marie JP, Mespoulhès-Rivière C, Ducharme NG. Modified first or second cervical nerve transplantation technique for the treatment of recurrent laryngeal neuropathy in horses.. Equine Vet J 2018 Jul;50(4):457-464.
    doi: 10.1111/evj.12788pubmed: 29193393google scholar: lookup
  6. Cercone M, Jarvis JC, Ducharme NG, Perkins J, Piercy RJ, Willand MP, Mitchell LM, Sledziona M, Soderholm L, Cheetham J. Functional electrical stimulation following nerve injury in a large animal model.. Muscle Nerve 2019 Jun;59(6):717-725.
    doi: 10.1002/mus.26460pubmed: 30815883google scholar: lookup
  7. Vanschandevijl K, Nollet H, Vonck K, Raedt R, Boon P, Roost D, Martens A, Deprez P. Functional electrical stimulation of the left recurrent laryngeal nerve using a vagus nerve stimulator in a normal horse.. Vet J 2011 Sep;189(3):346-8.
    doi: 10.1016/j.tvjl.2010.07.008pubmed: 20724182google scholar: lookup
  8. Cheetham J, Perkins JD, Jarvis JC, Cercone M, Maw M, Hermanson JW, Mitchell LM, Piercy RJ, Ducharme NG. Effects of Functional Electrical Stimulation on Denervated Laryngeal Muscle in a Large Animal Model.. Artif Organs 2015 Oct;39(10):876-85.
    doi: 10.1111/aor.12624pubmed: 26471139google scholar: lookup
  9. Martini P, Cercone M, Cheetham J, Koch KP. Experimental electrical field distribution measurements in a perfused ex vivo model. Biomed Tech 2012; 57(Suppl. 1):870–3.
    doi: 10.1515/bmt-2012-4147google scholar: lookup
  10. Langendorff O. Untersuchungen am überlebenden Säugethierherzen. Pflügers Arch 1895; 61:291–332.
    doi: 10.1007/BF01812150google scholar: lookup
  11. Berke G, Mendelsohn AH, Howard NS, Zhang Z. Neuromuscular induced phonation in a human ex vivo perfused larynx preparation.. J Acoust Soc Am 2013 Feb;133(2):EL114-7.
    doi: 10.1121/1.4776776pmc: PMC3562273pubmed: 23363190google scholar: lookup
  12. Berke GS, Neubauer J, Berry DA, Ye M, Chhetri DK. Ex vivo perfused larynx model of phonation: preliminary study.. Ann Otol Rhinol Laryngol 2007 Nov;116(11):866-70.
    doi: 10.1177/000348940711601113pubmed: 18074674google scholar: lookup
  13. Kruit AS, Smits L, Pouwels A, Schreinemachers MJM, Hummelink SLM, Ulrich DJO. Ex-vivo perfusion as a successful strategy for reduction of ischemia-reperfusion injury in prolonged muscle flap preservation - A gene expression study.. Gene 2019 Jun 15;701:89-97.
    doi: 10.1016/j.gene.2019.03.021pubmed: 30902788google scholar: lookup
  14. Blomkalns AL. Lactate–A Marker For Sepsis And Trauma. EMCREG 2006; 2:43–9.
  15. Karagiannis MH, Reniker AN, Kerl ME, Mann FA. Lactate measurement as an indicator of perfusion. Compend Contin Educ Vet 2006; 28:287–98.
  16. Chan FK, Moriwaki K, De Rosa MJ. Detection of necrosis by release of lactate dehydrogenase activity.. Methods Mol Biol 2013;979:65-70.
    doi: 10.1007/978-1-62703-290-2_7pmc: PMC3763497pubmed: 23397389google scholar: lookup
  17. Dave A, Maru L, Jain A. LDH (Lactate Dehydrogenase): A Biochemical Marker for the Prediction of Adverse Outcomes in Pre-eclampsia and Eclampsia.. J Obstet Gynaecol India 2016 Feb;66(1):23-9.
    doi: 10.1007/s13224-014-0645-xpmc: PMC4755940pubmed: 26924903google scholar: lookup
  18. Kim SS, Yang HW, Kang HG, Lee HH, Lee HC, Ko DS, Gosden RG. Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment.. Fertil Steril 2004 Sep;82(3):679-85.
    doi: 10.1016/j.fertnstert.2004.05.022pubmed: 15374714google scholar: lookup
  19. Weidemann A, Johnson RS. Biology of HIF-1alpha.. Cell Death Differ 2008 Apr;15(4):621-7.
    doi: 10.1038/cdd.2008.12pubmed: 18259201google scholar: lookup
  20. Kim JH, Bae HC, Kim J, Lee H, Ryu WI, Son ED, Lee TR, Jeong SH, Son SW. HIF-1α-mediated BMP6 down-regulation leads to hyperproliferation and abnormal differentiation of keratinocytes in vitro.. Exp Dermatol 2018 Nov;27(11):1287-1293.
    doi: 10.1111/exd.13785pubmed: 30230035google scholar: lookup
  21. Berchner-Pfannschmidt U, Frede S, Wotzlaw C, Fandrey J. Imaging of the hypoxia-inducible factor pathway: insights into oxygen sensing.. Eur Respir J 2008 Jul;32(1):210-7.
    doi: 10.1183/09031936.00013408pubmed: 18591338google scholar: lookup
  22. Wang GL, Semenza GL. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia.. Proc Natl Acad Sci U S A 1993 May 1;90(9):4304-8.
    doi: 10.1073/pnas.90.9.4304pmc: PMC46495pubmed: 8387214google scholar: lookup
  23. Wang GL, Semenza GL. Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction.. Blood 1993 Dec 15;82(12):3610-5.
    pubmed: 8260699
  24. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.. Proc Natl Acad Sci U S A 1995 Jun 6;92(12):5510-4.
    doi: 10.1073/pnas.92.12.5510pmc: PMC41725pubmed: 7539918google scholar: lookup
  25. Förster G, Arnold D, Bischoff SJ, Schubert H, Scholle HC, Müller AH. Laryngeal pacing in minipigs: in vivo test of a new minimal invasive transcricoidal electrode insertion method for functional electrical stimulation of the PCA.. Eur Arch Otorhinolaryngol 2013 Jan;270(1):225-31.
    doi: 10.1007/s00405-012-2141-1pubmed: 22875064google scholar: lookup
  26. Wu D, Yotnda P. Induction and testing of hypoxia in cell culture.. J Vis Exp 2011 Aug 12;(54).
    doi: 10.3791/2899pmc: PMC3217626pubmed: 21860378google scholar: lookup
  27. Sejersted OM, Hargens AR, Kardel KR, Blom P, Jensen O, Hermansen L. Intramuscular fluid pressure during isometric contraction of human skeletal muscle.. J Appl Physiol Respir Environ Exerc Physiol 1984 Feb;56(2):287-95.
    doi: 10.1152/jappl.1984.56.2.287pubmed: 6706739google scholar: lookup
  28. Ballard RE, Watenpaugh DE, Breit GA, Murthy G, Holley DC, Hargens AR. Leg intramuscular pressures during locomotion in humans.. J Appl Physiol (1985) 1998 Jun;84(6):1976-81.
    doi: 10.1152/jappl.1998.84.6.1976pubmed: 9609792google scholar: lookup
  29. Topalov I, Daskalov I. [Electrical stimulation for rapid assessment of extremity tissues vitality in critical ischemia].. Ann Chir 2004 Mar;129(2):79-82.
    doi: 10.1016/j.anchir.2003.12.010pubmed: 15050177google scholar: lookup
  30. Warther S, Sehner S, Raupach T, Püschel K, Anders S. Estimation of the time since death: post-mortem contractions of human skeletal muscles following mechanical stimulation (idiomuscular contraction).. Int J Legal Med 2012 May;126(3):399-405.
    doi: 10.1007/s00414-011-0665-3pubmed: 22245837google scholar: lookup
  31. Folkesson KT, Samuelsson A, Tesselaar E, Dahlström B, Sjöberg F. A human vascular model based on microdialysis for the assessment of the vasoconstrictive dose-response effects of norepinephrine and vasopressin in skin.. Microcirculation 2012 May;19(4):352-9.
    doi: 10.1111/j.1549-8719.2012.00170.xpubmed: 22332827google scholar: lookup
  32. Singh A, Laribi S, Teerlink JR, Mebazaa A. Agents with vasodilator properties in acute heart failure.. Eur Heart J 2017 Feb 1;38(5):317-325.
    doi: 10.1093/eurheartj/ehv755pubmed: 28201723google scholar: lookup
  33. Friebe M, Stahl J, Kietzmann M. The isolated perfused equine distal limb as an ex vivo model for pharmacokinetic studies.. J Vet Pharmacol Ther 2013 Jun;36(3):292-7.
    doi: 10.1111/jvp.12001pubmed: 22913456google scholar: lookup
  34. Dhein S, Giessler C, Heinroth-Hoffmann I, Leineweber K, Seyfarth T, Brodde OE. Changes in alpha(1)-adrenergic vascular reactivity in monocrotaline-treated rats.. Naunyn Schmiedebergs Arch Pharmacol 2002 Feb;365(2):87-95.
    doi: 10.1007/s00210-001-0515-9pubmed: 11819025google scholar: lookup
  35. Moritz A, editor. Klinische Labordiagnostik in der Tiermedizin. 7th ed. Stuttgart: Schattauer; 2014.
  36. Muñoz A, Riber C, Santisteban R, Lucas RG, Castejón FM. Effect of training duration and exercise on blood-borne substrates, plasma lactate and enzyme concentrations in Andalusian, Anglo-Arabian and Arabian breeds.. Equine Vet J Suppl 2002 Sep;(34):245-51.
    doi: 10.1111/j.2042-3306.2002.tb05427.xpubmed: 12405695google scholar: lookup
  37. Deschene K, Céleste C, Boerboom D, Theoret CL. Hypoxia regulates the expression of extracellular matrix associated proteins in equine dermal fibroblasts via HIF1.. J Dermatol Sci 2012 Jan;65(1):12-8.
    doi: 10.1016/j.jdermsci.2011.09.006pubmed: 21999945google scholar: lookup
  38. Pawlak EA, Geor RJ, Watts MR, Black SJ, Johnson PJ, Belknap JK. Regulation of hypoxia-inducible factor-1α and related genes in equine digital lamellae and in cultured keratinocytes.. Equine Vet J 2014 Mar;46(2):203-9.
    doi: 10.1111/evj.12092pubmed: 23663159google scholar: lookup
  39. Martano M, Altamura G, Power K, Restucci B, Carella F, Borzacchiello G, Maiolino P. Evaluation of Hypoxia-Inducible Factor-1 Alpha (HIF-1α) in Equine Sarcoid: An Immunohistochemical and Biochemical Study.. Pathogens 2020 Jan 14;9(1).
    doi: 10.3390/pathogens9010058pmc: PMC7168668pubmed: 31947661google scholar: lookup
  40. Hadjipanayi E, Schilling AF. Hypoxia-based strategies for angiogenic induction: the dawn of a new era for ischemia therapy and tissue regeneration.. Organogenesis 2013 Oct 1;9(4):261-72.
    doi: 10.4161/org.25970pmc: PMC3903695pubmed: 23974216google scholar: lookup
  41. Patan B, Budras KD, Licka TF. Effects of long-term extracorporeal blood perfusion of the distal portion of isolated equine forelimbs on metabolic variables and morphology of laminar tissue.. Am J Vet Res 2009 May;70(5):669-77.
    doi: 10.2460/ajvr.70.5.669pubmed: 19405908google scholar: lookup
  42. Otto S, Tast V, Michler JK, Mülling CK. A Case Study for a New Approach of a Constant Pressure Perfused Ex-Vivo Model of the Equine Larynx.. Biomed Tech (Berl) 2013 Aug;58 Suppl 1.
    doi: 10.1515/bmt-2013-4024pubmed: 24042603google scholar: lookup

Citations

This article has been cited 0 times.
Subscribe to our newsletter
Join 99,344 horse owners like you who receive our email updates on horse health and nutrition.