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Gardner Masters 4.20.15.pdf (1.96 MB)

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Lamellar Mitogen-Activated Protein Kinase and Hypoxia Signaling in a Sepsis-Related Laminitis Model and a Novel Supporting Limb Laminitis Model

Gardner, Alison
http://rave.ohiolink.edu/etdc/view?acc_num=osu1429120162

Abstract Details

2015, Master of Science, Ohio State University, Comparative and Veterinary Medicine.
The term laminitis refers to dysadhesion of the lamellar basilar epithelial cell (LBEC) from the basement membrane, resulting in distal displacement of the third phalanx. Multiple etiologies of laminitis exist, which can be grouped into three broad categories: equine metabolic syndrome-associated laminitis, sepsis-related laminitis (SRL) and supporting limb laminitis (SLL). This thesis centers on two different laminitis models, the first being based on sepsis-related laminitis and the second being a novel model for supporting limb laminitis. Sepsis-related laminitis, an often fatal sequela in critical equine patients secondary to endotoxemia and sepsis-potentiating diseases, appears to be due to aberrant laminar cell signaling reportedly involving inflammatory and possibly other signaling pathways. The only documented effective treatment for sepsis-related laminitis is regional deep hypothermia (RDH, foot submerged in ice water). Mitogen-activated protein kinases (MAPKs), activated in inflammation or downstream of growth factor signaling, are potential therapeutic targets for many disease processes. Our objectives were to assess MAPK signaling in laminar tissue in a model of sepsis-related laminitis and to determine the effect of RDH on MAPK signaling. Lamellar concentrations of MAPKs were assessed from two groups of horses receiving a carbohydrate-overload with samples collected at different time points versus a control. Another set was taken from a carbohydrate-overload model where one front limb was treated with RDH while the other remained at ambient temperature. Lamellar concentrations and cellular localization of the MAPKs and the signaling proteins of the interconnected protein kinase B (Akt)/Phosphoinositide 3 kinase (PI3k)/mammalian target of rapamycin (mTOR) pathway were assessed. Whereas no change in lamellar p38 MAPK was found in the CHO models, lamellar concentrations of growth factor-related signaling molecules including the phosphorylated/activated MAPK, extracellular signal-regulated kinase (ERK) 1/2 and its downstream effector ribosomal protein S6 (RPS6) were increased (p<0.05) at the onset of laminitis, as was the MAPK stress-activated protein kinase/c-jun N terminal (SAPK/JNK) 1/2, the cellular negative-energy balance signaling protein AMP-activated protein kinase a (AMPKa), and Akt. Hypothermia did not inhibit ERK 1/2, but did cause a decrease in RPS6 phosphorylation/activation but an increase in SAPK/JNK 1/2 phosphorylation/activation. Signaling related to growth factor-related pathways should be further investigated in SRL, especially since it has been implicated in cell-cell dysadhesion and upregulation of the ERK 1/2 pathway. The pathophysiology of equine supporting limb laminitis (SLL), a common and often fatal complication of equine orthopedic disease, is poorly understood. Often horses survive the initial catastrophic trauma, e.g. fracture, only to succumb to fulminant failure of the contralateral limb. Suggested causes of lamellar failure include inflammatory injury, hypoxia and mechanical strain. We hypothesized that lamellar hypoxia occurs in the supporting limb (SL) resulting in increase in lamellar hypoxia-inducible factor-1a (HIF-1a). A novel model of SLL was used in this study in which a custom shoe insert causing instability to the sole surface upon weight bearing was placed on one forelimb of horses resulting in excessive weighting of the contralateral forelimb (SL). Lamellae were harvested and immediately snap-frozen from all four limbs 48 h post-application of the shoe. Western immunoblotting and real time-quantitative PCR (qPCR) were used to assess markers of hypoxia (HIF-1a) inflammation and stretch. The only change noted was an increase (P<0.05) in lamellar HIF-1a protein concentrations in the SL compared to one hindlimb and a trending increase in the other. Genes including those indicated in stretch, metabolism and inflammation were not upregulated, nor was HIF-1a mRNA. These results indicate that lamellar hypoxia and HIF-1a may play a central role in SLL and future research should focus on therapeutic options, including novel shoeing options on the SL which may allow increased vascular oxygen delivery to the distal limb. HIF-1a may have value as a biomarker of lamellar hypoxia and be used to assess the efficacy of SLL treatments. The two models (CHO and SLL) exhibit differences between the arms of sepsis-related and supporting limb laminitis. However, further work must be done on cellular growth factor signaling and metabolic pathways, as recent evidence shows that while SRL and SLL have differing properties in MAPK, Akt/PI3k/mTOR, and HIF-1a activation as well as pro-inflammatory cyotokine gene expression, RPS6 may play a role in dysadhesion of the epidermal lamellae.
James Belknap, DVM, PhD (Advisor)
Teresa Burns, DVM, PhD (Committee Member)
Prosper Boyaka, PhD (Committee Member)
Ramiro Toribio, PhD (Committee Member)
100 p.
Cellular Biology; Veterinary Services
equine, laminitis, sepsis, supporting limb, mitogen-activated protein kinase, hypoxia

Recommended Citations

Citations

  • Gardner, A. (2015). Lamellar Mitogen-Activated Protein Kinase and Hypoxia Signaling in a Sepsis-Related Laminitis Model and a Novel Supporting Limb Laminitis Model [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429120162

    APA Style (7th edition)

  • Gardner, Alison. Lamellar Mitogen-Activated Protein Kinase and Hypoxia Signaling in a Sepsis-Related Laminitis Model and a Novel Supporting Limb Laminitis Model. 2015. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1429120162.

    MLA Style (8th edition)

  • Gardner, Alison. "Lamellar Mitogen-Activated Protein Kinase and Hypoxia Signaling in a Sepsis-Related Laminitis Model and a Novel Supporting Limb Laminitis Model." Master's thesis, Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429120162

    Chicago Manual of Style (17th edition)

osu1429120162
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This open access ETD is published by The Ohio State University and OhioLINK.