Evaluation of the Effects of Therapeutic Digital Hypothermia on Lamellar Signaling
in Sepsis Related Laminitis
Thesis
Presented in Partial Fulfillment of the Requirements for the Degree of Masters of Science
in the Graduate School of The Ohio State University
By
Kathryn V. Dern, B.S., D.V.M.
Graduate Program in Comparative and Veterinary Medicine
The Ohio State University
2017
Master’s Examination Committee:
Professor James K. Belknap, Advisor
Assistant Professor Teresa Burns
Associate Professor Estelle Cormet-Boyaka
Professor Prosper N. Boyaka
Copyrighted by
Kathryn Virginia Dern
2017
Abstract
Laminitis is a debilitating, often fatal disease of the equine foot which, specifically, refers to the loss of integrity of the digital lamellae, commonly termed the suspensory apparatus of the distal phalanx. Causes of laminitis can be separated into three broad categories: sepsis related laminitis, equine metabolic syndrome associated laminitis, and supporting limb laminitis. Although the anamneses of patients afflicted by these disparate types of laminitis can be varied, many similarities can be drawn between the ultrastructural changes seen in the lamellar tissue of the affected animals: namely, stretching of the lamellar basal and parabasal epithelial cells, loss of hemidesmosomal integrity in the basal epithelial cells, and lengthening of the secondary epidermal lamellae.
Recent investigations into the pathogenesis of sepsis related laminitis have implicated inflammatory signaling as a causative agent for the lamellar damage seen in acute sepsis related laminitis, and, more importantly, these investigations have discovered a decrease in lamellar inflammatory signaling with the institution of therapeutic digital hypothermia. Therapeutic digital hypothermia is the only therapy demonstrated to not only treat but prevent the lamellar damage documented to occur in sepsis related laminitis. Therefore, the aims of the first two investigations outlined in this thesis were to characterize the effect of digital hypothermia instituted after the onset of lameness on
ii gene expression of inflammatory mediators in the oligofructose model of sepsis related laminitis.
Interestingly, both experiments 1 and 2 demonstrated that, although the histopathology scores demonstrated profound cryoprotection of the lamellar tissue treated with digital hypothermia, this protection did not correlate with a decrease in lamellar inflammatory signaling. These data indicated that the increase in lamellar inflammation long noted to be present in acute sepsis related laminitis may not be the causative event leading to structural failure of the digital lamellae.
Since inflammatory signaling was determined unlikely to be the source of the cryoprotective effects of digital hypothermia, growth factor signaling pathways recently investigated in the euglycemic-hyperinsulinemic clamp model of EMSAL were investigated in the oligofructose model of sepsis-related laminitis. Notably, not only were there increases in phosphorylation (indicating activation) of the same downstream effectors of growth factor signaling pathways phosphorylated in sepsis related laminitis as in EMSAL, treatment with digital hypothermia inhibited phosphorylation of these same downstream effector proteins. Thus, experiment 3 serves not only to implicate growth factor signaling pathways in the pathogenesis of sepsis related laminitis (thus establishing a pathophysiological connection between SRL and EMSAL), but also reveals a correlation between a decrease in the activation of growth factor signaling pathways and the cryoprotection conferred by therapeutic digital hypothermia.
iii
Acknowledgements
I would like to thank Dr. Jim Belknap for his guidance and training both in the labarotory and on the clinic floor; his patience and encouragement are much appreciated.
Equally, I would like to thank Mauria Watts for her perfectionism and attention to detail in every aspect of laboratory work and manuscript preparation; I felt truly spoiled to have the opportunity to pick her brain at will. I would also like to thank the members of my masters committee, Dr. Burns, Dr. Boyaka, and Dr. Cormet Boyaka for their involvement with my masters project.
I also need to express my appreciation for the technicians and residents of the
Equine Surgery and Medicine services: it has been a privilege to work with such a dynamic group of women for the past 3 years…you will be missed.
Finally, I would like to thank my husband, Eric, for never questioning what was so important about horse feet (or throats, or colons), and for ensuring that our pets survived two internships and a residency.
iv
Vita
2004…………………………………….……….…..Cedarcrest High School, Duvall, WA
2008……………….…B.S., Animal Science, Washington State University, Pullman, WA
2012…………………………………D.V.M, Colorado State University, Fort Collins, CO
2012-2013…………………….…..Intern, Peterson and Smith Equine Hospital, Ocala, FL
2013-2014……………………..Intern, Rood and Riddle Equine Hospital, Lexington, KY
2013-2016………...Resident, Equine Surgery, The Ohio State University, Columbus, OH
Fields of Study
Major Field: Comparative and Veterinary Medicine
v
Table of Contents
Abstract……………………………………………………………………………………ii
Acknowledgements……………………………………………………………...……...... iv
Vita………………………………………………………………………………………...v
List of Figures……………………………………………………………………..…….viii
List of Tables……………………………………………………………………………..ix
Chapter 1: Introduction……………………………………………………………………1
1.1: Laminitis: Anatomical Considerations ………………………………………………1
1.2: Laminitis: Pathophysiology ………………………………………………….……....8
1.3: Sepsis Related Laminitis ……...…………………………………………………….12
1.4 Inflammation and Laminitis …………………………………………………………15
1.5 Laminitis Treatment: Hypothermia …………………………..…………………...…17
Chapter 2: Effect of Delayed Digital Hypothermia on Lamellar Inflammatory Signaling in the Oligofructose Laminitis Model ………………………..………………….………21
2.1 Introduction………….……………………………………………………………….21
2.2 Materials and Methods ………………………………………………………………22
2.3 Results…..…………………………………………………………………..………..26
2.4 Discussion……………………………………………………………………………34
vi Chapter 3: Controlled Digital Hypothermia in Sepsis Related Laminitis at Clinically
Relevant Timepoints …………………………………………………………………….39
3.1 Introduction………….……………………………………………………………….39
3.2 Materials and Methods ………………………………………………………………41
3.3 Results….…………………………………………………………………………….46
3.4 Discussion…………………………………………………………………………....53
Chapter 4: Growth Factor Signaling in Sepsis Related Laminitis……………………….58
4.1 Introduction…………………………………………………………………………..58
4.2 Materials and Methods……………………………………………………………….59
4.3 Results………………………………………………………………………………..61
4.4 Discussion…………………………………………………………………………....67
Chapter 5: Discussion……………………………………………………………………71
5.1 Inflammation and Sepsis-Related Laminitis…………………………………………71
5.2 Growth Factor Signaling and Laminitis ...... 73
References………………………………………………………………………………..80
vii
List of Figures
Figure 1 Anatomy of the Equine Hoof Capsule….…………………………………….....3
Figure 2 Cellular Structure of the Dermal/Epidermal Junction……………………….….3
Figure 3 Phosphorylation of Growth Factor Signaling Pathways……………………….12
Figure 4 Scatter plots of lamellar mRNA concentrations of inflammatory molecules…29
Figure 5 Lamellar mRNA concentrations of inflammatory molecules: Archived non- septic controls (CON) vs OG3 vs OG1…………………………..…………..………….32
Figure 6 Assessment of lamellar leukocyte concentration……………………..………..52
Figure 7 Western blots exhibiting phosphorylation of STAT3 (Y705) and STAT3 (S727)
……………………………………………………………………………………………64
Figure 8 Western blots exhibiting phosphorylation of RPS6……………………………65
Figure 9 Western blots exhibiting phosphorylation of p70S6K…………………………65
Figure 10 Immunohistochemistry comparing phosphorylation of RPS6 of AMB OF vs
ICE OF limbs……………………………………………………………………………66
Figure 11 Immunohistochemistry comparing phosphorylation of STAT3 of AMB OF vs
ICE OF limbs……………………………………………………………………………66
Figure 12 Representation of the phosphorylation (activation) of downstream effectors of mTORC1 in SRL……………………………………………………………………….74
Figure 13 Representation of the lack of phosphorylation (activation) of downstream effectors of mTORC1 in limbs subject to digital hypothermia…………………………74
viii
List of Tables
Table 1 Lamellar tissue inflammatory mediatory copy number expression data…….….28
Table 2 Statistical correlation of lamellar mRNA concentrations of specific mediators with the histological scores….…………………………………………………………...30
Table 3 Lamellar tissue inflammatory mediator copy number expression data: control
(CON) compared with untreated (NON-CRYO) limbs in subjects in which tissue was harvested at OG1 and 36 hours after onset of OG1……………………………..……….33
Table 4 LSMeans of the copy number data predicted using each model ……………….50
ix
Chapter 1: Introduction
1.1 Laminitis: Anatomical Considerations
The first record of laminitis in equine veterinary writings dates back to 330 BC, when Aristotle described “foot-ill” in which “the casting of the one hoof takes place simultaneously with the growing of the other hoof underneath.”1 Since then, over 50 potential etiologies ranging from the exceedingly simple (avocado consumption) to more complex (interruption of methionine keratin synthesis) have been proposed as the causative agent for this devastating disease which still shortens the working life and sometimes results in the euthanasia of 2-7% of the equine population2. Despite considerable international research effort, the exact pathways leading to lamellar damage have yet to be elucidated3,4. An understanding of the causes and potential treatment of laminitis requires a detailed understanding of the structure of the suspensory apparatus of the distal phalanx and the levels at which it can fail.
The main weight bearing structure of the equine hoof is the hoof wall, which is connected to the appendicular skeleton by the suspensory apparatus of the distal phalanx. The suspensory apparatus of the distal phalanx serves to redirect the weight- bearing forces of the horse (transmitted through the appendicular skeleton and ending on the third phalanx), to the hoof wall, and thus to the ground. Forces generated upon the distal phalanx relayed through this unique structure include compressive forces due to the body weight of the animal, tensile forces on the dorsal surface due to the action
1 of the common digital extensor tendon, tensile forces on the palmar surface due to the action of the deep digital flexor tendon, and compressive forces generated against the sole5. The successful absorption and transmission of these forces by the third phalanx depends on compliant nature of the suspensory apparatus of the distal phalanx. The suspensory apparatus of the distal phalanx is composed of the stratum lamellum, a specialized epidermal layer of interdigitating primary and secondary epidermal and dermal lamellae (Figure 1). Unique to the equine hoof, the layer of secondary epidermal lamellae acts to increase the weight-bearing surface area of each hoof to a staggering 0.8m2, approximately the surface area of the skin of a small human6. This increase in surface area allows each hoof to bear peak forces up to 3x body weight at the gallop; making the suspensory apparatus of the distal phalanx an essential characteristic of the equine athlete5. During weight bearing and locomotion, the forces generated by the weight of the horse and the opposing ground reaction force transmitted via the hoof wall exert large distractive forces on the interdigitation of the secondary epidermal and dermal lamellae, with the basement membrane connecting the dermal and epidermal components playing a pivotal role7-9. The linkage between the lamellar basal epithelial cell and the basement membrane is a network of dense adhesion plaques known as hemidesmosomes9. Failure of this attachment of the lamellar basement epithelial cell to the basement membrane, or even loss of the cytoskeletal integrity of the epithelial cell due to stretching and thus loss of the connection between the hoof capsule and distal phalanx, destroys the integrity of the suspensory apparatus of the distal phalanx and allows for the distal displacement of the distal phalanx within the hoof capsule: a hallmark lesion of laminitis9-11 (Figure 2).
2 Figure 1: The structure of the equine distal phalanx and relationship to the hoof capsule. Inset: Interdigitation of the primary and secondary dermal and epidermal lamellae to form the stratum lamellum.
Figure 2: Cellular structure of the dermal/epidermal junction.
3 The clinical signs associated with distal displacement of the distal phalanx within the hoof capsule were first assigned grades by Obel in 1948. Obel Grade 1
(OG1) laminitis describes a patient/study subject which shifts its feet fairly constantly
(hence the use of pedometers to objectively grade laminitis in study subjects), and has a detectable lameness at the trot. Obel Grade 2 (OG2) laminitis describes a patient which is appreciably lame at the walk. Obel Grade 3 (OG3) lameness is an animal that is reluctant to bear weight on one forelimb if the other is lifted off of the ground, and
Obel Grade 4 (OG4) laminitis describes an animal that is reluctant to move and adapts the classic sawhorse stance to distribute as much weight as possible to its hindlimbs12.
In a manuscript assessing basement membrane pathology in acute equine laminitis in
1996, Pollitt developed a grading system for the histopathological changes observed in the laminitic patient which was found to have a strong correlation (r2=0.94) with the
Obel grades. In his description, Grade 1 was assigned to lamellar tissue in which there was rounding of the basal cell nuclei and elongation of the secondary epidermal lamellae, with some separation of the basement membrane away from the epidermal cells at the tips of the lamellae. Grade 2 histopathological lesions included absence of basement membrane at the tips of the lamellae, and progression into Grade 3 was indicated by complete separation of the primary epidermal and dermal lamellae13. The strong correlation between these histopathological scores and the Obel grades (based on clinical signs) is vital to interpretation of most current laminitis studies, since the study subjects are administered continuous perineural regional anesthesia, preventing accurate assessment of lameness late in the study period.
4 The strong correlation between the clinical signs of laminitis and the histopathological changes is also important because deeper investigation into the dermal/epidermal junction at the cellular level has provided key insight into potential pathophysiologies of acute laminitis. Importantly, French et al were able to demonstrate that the dose of oligofructose used in a CHO model of sepsis related laminitis (SRL) correlated with hemidesmosome and basement membrane zone damage. Namely, with an increase in OF dose, the nuclei of the LBEC became more rounded and were situated more closely to the cell base and basement membrane zone, indicating loss of integrity of the intermediate filament cytoskeleton which keeps the nucleus appropriately oriented within the cell. Additionally, the cell cytoplasm took up less stain, and tonofilaments (intermediate filaments) were noted to be no longer adjacent to the HD, but rather laying in wavy bundles along the cell nucleus. This manuscript served to significantly provide a link between the histopathologic changes observed in OF-induced laminitis (already correlated to the Obel grades of laminitis by
Pollitt in 1996) to a decrease in the number of hemidesmosomes and compromise of the cytoskeletal integrity of the LBEC13,14.
When viewed under transmission electron microscopy (TEM), the basement membrane is mainly composed of a lamina densa, which is adhered to the lamellar basal epithelial cell by numerous electron dense adhesion plaques or hemidesmosomes
(HD)9. Mammalian species have two types of hemidesmosomes: classical Type 1, which are found in complex epithelia such as the skin, bladder, and lamellar tissue, and
Type II which are found in the intestine.
5 The adhesion complexes between the LBEC and the basement membrane are composed of Type 1 hemidesmosomes, which are best understood through the roles of their 5 structural proteins: integrin α6 β4, BPAG2 (BP180), plectin isoform 1a (P1a), tetraspanin CD151, and bullous pemphigoid antigen 1 isoform e (BPAG1e or BP230) .
At the dermal/epidermal junction in equine lamellae, these 5 proteins act in concert to connect the intermediate filament cytoskeleton of the LBEC to the anchoring filaments of the basement membrane. Integrin α6 β4 is one of two transmembrane components of the hemidesmosome (the other is BPAG2); and in addition to its expression in epithelial cells, it can also be found in endothelial cells, astrocytes, neurons, and
Schwann cells. The cytoplasmic domain of integrin α6 β4 interacts with the keratin intermediate filaments of the LBEC cytoskeleton, and the extracellular domain acts as a receptor for laminin, a major glycoprotein component of the anchoring filaments of the BM15,16. The importance of integrin α6 β4 in the stability of the dermal/epidermal junction is highlighted by the severe skin fragility and blistering (epidermolysis bullosa) observed in both humans with mutations in either the α6 or β4 subunits, and in integrin α6 knockout mice17. Immunofluorescence labelling of integrin α6 in equine lamellar tissue outlined the lamellar basal epithelial cell of the secondary epidermal lamellae, effectively documenting its presence in the hemidesmosome/adhesion complexes of equine lamellar tissue18.
The other transmembrane receptor of the HD, BPAG2 (BP180) connects to the outer plaque of the hemidesmosome, spans the lamina lucida, and has a tail which resides in the lamina densa of the basement membrane. Although mutations in the gene coding for BP180 lead to junctional epidermolysis bullosa in humans, the action of
6 BP180 alone binding to the basement membrane in the absence of integrin α6 β4 was not strong enough to mediate the adhesion of epithelial cells to the basement membrane in animal models, suggesting a more significant role of integrin α6 β4 in hemidesmosome structure and stability15,19. As with integrin α6 β4, Pollitt et al were able to use immunofluorescent labelling to demonstrate the presence of BP180 in the
LBEC, localized adjacent to the basement membrane18.
While integrin α6 β4 and BP180 act to connect the hemidesmosome to the basement membrane, plectin isoform 1a (P1a) and bullous pemphigoid antigen 1 isoform e (BPAG1e or BP230) serve to anchor the hemidesmosome to the intermediate filament cytoskeleton of the epithelial cell. Binding to integrin α6 β4 on the cytoplasmic face of the hemidesmosome, plectin has a tripartite structure which is able to bind with a wide array of cellular components (tight junctions, focal adhesions, costamers, intermediate filaments, and mitochondrial membranes), making it a key player in epithelial cell structure and the intermediate filament anchorage sites within the cytoplasm of cells. The importance of P1a in hemidesmosomal integrity is underscored by investigations of the skin blistering disease EBS-Ogna in human patients, in which a dominant mutation leads to the cleavage of the HD-P1a association, resulting in marked skin fragility in response to mechanical stress20. The second protein critical to the hemidesmosome-IF connection is bullous pemphigoid antigen 1 isoform e (BPAG1e or BP230), which binds to the HD components integrin
α6 β4, BP 180, and keratins composing the intermediate filaments15. As with the transmembrane components of hemidesmosomes, presence of P1a (plectin) and BP230 was demonstrated in the LBEC using immunofluorescence, effectively confirming that
7 the hemidesmosomes anchoring the lamellar basal epithelial cell to the basement membrane in equine lamellar tissue share similar characteristics to human epithelial/epidermal hemidesmosomes18.
Interestingly, the cytoskeletal derangements observed in the LBEC of a laminitic patient are also seen in the epithelial to mesenchymal transition of the early stages of neoplasia, namely, the loss of cell-cell adhesion structure components (HDs or junctions) and the subsequent loss of normal cytoskeletal structure21. Although equine lamellar basal epithelial cells obviously do not progress to the metastatic and invasive stages seen with carcinomas, the pathophysiology of the loss of the initial cell- cell adhesion and detachment from the basement membrane bears similarity worth noting, especially because growth factor signaling patterns demonstrated in recent laminitis models (both EHC and CHO) have also been demonstrated in human oral carcinoma and other neoplasias22-26.
1.2 Laminitis: Pathophysiology
Modern investigations of laminitis divide the disease process into 3 major subtypes based on inciting cause: 1)sepsis-related laminitis (SRL), 2) endocrinopathic laminitis (also known as equine metabolic syndrome associated laminitis or EMSAL), and 3) supporting limb laminitis (SLL), all of which share the same common outcome: distal displacement of the distal phalanx within the hoof capsule. Investigations into the ultrastructural changes which occur with both sepsis-related and endocrinopathic laminitis focus on the secondary epidermal lamellae (SEL); severe laminitis is characterized on a histological level by extremely elongated SELs with a detached basement membrane13,14. Since the lamellar basal epithelial cell (LBEC) of the
8 secondary epidermal lamella is anchored to the basement membrane by hemidesmosomes, it is not surprising that loss of hemidesmosomal integrity (a decrease in the number of hemidesmosomes) was observed in experimental models of both sepsis-related and endocrinopathic laminitis11,14,27. Although the three main subsets of laminitis (SRL, EMSAL, and SLL) have a common endpoint, there are differences between their etiologies and pathogeneses which will be briefly reviewed.
Supporting Limb Laminitis
Supporting limb laminitis (SLL) is arguably the most fatal form of laminitis, with a 50% mortality rate and a 12 % incidence in patients requiring full limb, half limb, or transfixation pin casts28,29. Although SLL can occur secondary to any unilateral non-weightbearing lameness, risk factors for SLL include heavier body weight, longer period of time in a cast, and the use of a full limb or transfixation pin cast29. Historically, the lack of an adequate model for induction of SLL has prevented controlled investigations into its pathophysiology. Recently, however, the development of a shoe with a custom V insert which effectively mimics a severe unilateral forelimb lameness, thus increasing weight-bearing on the contralateral limb, was used to evaluate hypoxia induced signaling in lamellar tissue30. Further controlled investigations using this model will allow for the further characterization of the pathophysiology of SLL.
Equine Metabolic Syndrome Associated Laminitis (EMSAL)
Rather than describing a specific disease process, the term Equine Metabolic
Syndrome indicates a cluster of clinical signs and characteristics which is recognized to place a patient at an increased risk for developing laminitis. Although there may be
9 multiple mechanisms at play in the development of laminitis in the EMSAL patient, the recognition of hyperinsulinemia as a risk factor for pasture associated laminitis (a subcategory of EMSAL), as well the ability to induce laminitis using supraphysiologic concentrations of insulin, have led to the belief that insulin dysregulation plays a significant role in the development of laminitis in the EMSAL patient 31-33.
EMSAL laminitis is induced experimentally through the use of the euglycemic- hyperinsulinemic clamp model (EHC), which has been shown to induce laminitis within 48 hours in study subjects. Furthermore, the lamellar tissue of animals subject to the EHC model exhibits the same changes observed in naturally occurring EMSAL: 1) elongation of the SEL (proposed to be due to cellular stretching), and 2) a mild amount of dysadherence of the LBEC from the basement membrane (specifically at the tips of the SEL which are located on the axial PEL)11,32,34,35. The establishment of the euglycemic-hyperinsulinemic clamp as a model for EMSAL has allowed for more rigorous investigation of the cellular signaling pathways responsible for these ultrastructural changes in the laminitic tissue.
Although a hyperinsulinemic state induces laminitis in experimental models, immunohistochemistry has revealed a lack of insulin receptors on lamellar basal epithelial cells and an extensive distribution of insulin-like growth factor receptors on lamellar epithelium in the same study subjects36. This shifted investigative focus away from insulin receptors to the activity of insulin-like growth factor receptors (IGF-1R) in the development of EMSAL. A subsequent study demonstrated the phosphorylation of the downstream effectors of IGF-1R in study subjects in which laminitis was induced using the euglycemic-hyperinsulinemic clamp model37(Figure 3). IGF-1R
10 modulated pathways have also been investigated in tumor biology, in which the characteristics of a cell undergoing epithelial to mesenchymal transition (including cytoskeletal dysregulation and dysadherence from the basement membrane) are similar to the ultrastructural changes which occur in the lamellar basal epithelial cell in
EMSAL33,38-42. IGF-1R pathways are reported to be well conserved amongst mammalian species, and result in the phosphorylation of two separate pathways: the
MAPK and P13K cellular signaling cascades. A recent manuscript investigating the
IGF-1R pathway in the EHC model of EMSAL demonstrated significantly increased phosphorylation of ERK 1/2 and P90RSK (MAPK pathway), Akt and p70S6K (P13K pathway), and RPS6 (converging point of MAPK and P13K pathways) in animals subjected to the EHC model when compared to control study subjects37. These data suggest that the cytoskeletal dysregulation, elongation/stretching of the secondary epidermal lamellae, decrease in hemidesmosomes, and separation from the basement membrane seen in the EHC model of EMSAL, and in EMSAL patients, may be due to cellular signaling events downstream of the IGF-1R.
11 Figure 3: Representation of phosphorylation (activation) of multiple downstream effectors of growth factor signaling pathways in EMSAL.
1.3 Sepsis Related Laminitis
Although EMSAL likely represents a greater percentage of chronic laminitis cases than SRL, laminitis in adult horses is most classically associated with endotoxemia and sepsis. Sepsis related laminitis (SRL) is a complication of a wide array of clinical conditions such as pleuropneumonia, metritis, large colon volvulus, or colitis and has been induced experimentally through the use of the starch overload model, black walnut extract model, and, most recently, oligofructose bolus model. As with any experimental models, the goal is to most closely emulate the clinical patient.
The starch overload model (administration of a mixture of 85% cornstarch and 15%
12 wood flour via nasogastric intubation) induces laminitis within 32-48 hrs in treated animals, mirroring the clinical timeline of laminitis development post endotoxemic event seen in clinical patients43. A weakness of the starch overload model, however, is the fact that 20-30% of treated animals do not develop laminitis secondary to administration.
In an attempt to induce SRL with a model more closely mimicking the rapid consumption of lush grass, the oligofructose laminitis induction model was developed.
After administration of a 10g/kg bolus of oligofructose via nasogastric intubation,
100% of study subjects progress to clinical laminitis with identical histopathological changes and a similar lameness profile to both CHO overload and naturally occurring
SRL cases27,44. Importantly, the OF model allowed for more reliable experimental induction of SRL, contributing to a rapid increase in the body of research investigating
SRL pathophysiology.
The observation that naturally occurring cases of laminitis could be seen after ingestion of black walnut shavings led to the use black walnut extract (BWE) as an experimental model, with the perceived advantage that the study subjects do not develop systemic illness to the degree of the starch overload or OF induction models45-
47. Although the BWE model is able to induce signs of systemic inflammation including hemodynamic alterations, leukocyte emigration, and an increase in inflammatory signaling, it does not seem to cause the same changes in cecal microflora as seen in the starch overload and OF induction models48-54. The starch overload and
OF SRL induction models cause a decrease in cecal pH and subsequent death and lysis of bacteria, resulting the release of their cell wall components and DNA material, loss
13 of mucosal barrier integrity, and systemic circulation of these toxic components52-54.
This, in turn, triggers a systemic inflammatory response syndrome, of which SRL is a known sequelae. Since BWE study subjects do not seem to undergo prolonged illness and SIRS, their lamellar tissue is not subject to the same sustained inflammatory insult as the clinical SRL patient45,55. For this reason, while BWE is able to induce laminitis in study subjects, its lack of close correlation with the clinical SRL patient has relegated its use to investigation of early lamellar inflammatory events45.
With the establishment of both starch overload and OF bolus as robust models of naturally occurring SRL, the pathophysiology and treatment of SRL has received a great degree of research attention. In the clinical patient, SRL is often secondary to colitis, pleuropneumonia, or any other morbidity which leads to release of pathogen associated molecular patterns (PAMPS), damage associated molecular patterns
(DAMPS), and subsequent activation of the host’s innate immune system56-58. Pro- inflammatory in nature, this immune response is designed to protect the host from microbial infection; however, if the inflammatory cascades go unchecked systemic inflammatory response syndrome (SIRS) can occur. Systemic inflammatory response syndrome (SIRS) is confirmed in the equine patient having at least 2 of the following criteria: abnormality in leukocyte count or distribution (leukopenia, leukocytosis, or
>10% band neutrophils), hyperthermia or hypothermia, tachycardia, or tachypnea59. In human sepsis, the epithelium and endothelium of the lungs, kidney, and brain are particularly susceptible to damage60,61. Early work in equine SRL demonstrated that several key processes, including leukocyte activation, endothelial activation, leukocyte adhesion and migration, and a profound inflammatory response which were the
14 hallmarks of damage in the human shock organs were also present in the damaged lamellar tissue from laminitic study subjects49,62,63. This led to the widespread consideration of lamellar tissue as a “shock organ” of the equine body, similar to the lungs, kidney, or brain of the human septic patient. This encouraged the comparison of the pathophysiology of end organ damage in human SIRS and MODS patients to the damage seen in the lamellar tissue of the equine septic patient, with the important difference that the human kidney and lungs are not subject to the extreme mechanical stresses placed on lamellar tissue.
Early laminitis research focused on hypoxia and decreased perfusion as causes for dysfunction of the lamellar basal epithelial cell64,65, but as evidence in the human medical literature began to mount that there was little chnage in blood flow in the
“target organs” of human sepsis, similar work in laminitis has demonstrated that increased blood flow was actually present in the acute stages of laminitis66-68. This has led to a recent shift in thought that the pro-inflammatory imbalance seen in both human and equine septic patients is responsible for the end-organ damage seen in the lamellae of horses and the lung, kidney, and brain of humans69.
1.4 Inflammation and Laminitis
Early work done in the BWE model of laminitis demonstrated first the increased expression of IL-1β (a pro-inflammatory cytokine central to the development of both human and equine sepsis) in lamellar tissue from treated study subjects70. This work was followed closely by demonstration of increased expression of cyclooxygenase-2, IL-6, and IL-8 in lamellar tissue from treated study subjects using the BWE model of laminitis71,72. These developments, coupled with the demonstration
15 of marked neutrophil emigration into the lamellar tissue of BWE study subjects, have led to strong support for inflammation as an agent associated with lamellar disruption in sepsis related laminitis63,73.
The first investigation into inflammatory signaling in the CHO overload model revealed that, as in BWE models of SRL, there was an increase in expression of IL-1β,
IL-6, IL-8, COX-2, and the leukocyte adhesion molecules ICAM-1 and E-selectin in the laminitic study subjects at the onset of OG1 lameness74. This study also investigated inflammatory signaling at two separate timepoints: prior to the onset of clinical lameness (DEV) and at the onset of OG1 laminitis (an increase in shifting back and forth on the front feet). This subcategorization of study subjects led to the discovery that there is a marked difference in the temporal patterns of inflammation between the BWE and CHO models of SRL. In the CHO study, the greatest peak in gene expression of inflammatory mediators was seen in lamellar tissue harvested at the
OG1 timepoint (at the time of clinically detectable lameness), whereas expression of inflammatory mediators peaked during the developmental stage of BWE-induced laminitis49,70,72,74. This discrepancy mirrors other animal models of organ damage post septic injury: the cecal ligation and puncture and endotoxin models. In the case of the cecal ligation and puncture (CLP) model, there is a delayed and prolonged inflammatory response, as was seen in the CHO model of SRL75,76. The endotoxin mouse model, however, incites a rapid systemic inflammatory response, similar to the
BWE model of SRL76. In both cases, the delayed and sustained model is preferred as it correlates more closely with the disease process in the clinical patient. Evaluation of the gene expression of inflammatory mediators in the oligofructose SRL model
16 revealed an expression pattern similar to the starch overload model: a delay in the increase in inflammatory mediators until the onset of clinical signs followed by a marked pro-inflammatory response77.
Interestingly, despite the robust inflammation present in the human septic patient, meta-analyses investigating the efficacy of three different methods of anti- inflammatory therapy (administration of glucocorticoids, anti-endotoxic therapy, and therapy targeting specific pro-inflammatory mediators) have all failed to demonstrate a decrease in the morbidity or mortality rate of human patients suffering from sepsis or septic shock78. This is similar to an investigation into the effects of ketoprofen, phenylbutazone, and flunixin meglumine on the apoptotic index of lamellar tissue from horses in which laminitis was induced using the starch overload model: there was no protective effect of NSAID administration on the lamellae when compared to saline controls79. Although this lack of efficacy is not surprising given the complex nature of the biological events surrounding sepsis and the degree of crosstalk between pro and anti-inflammatory pathways, it nonetheless underscores the need for more targeted investigations into the pathophysiology, at the molecular level, of the end organ damage in both human and equine sepsis.
1.5 Laminitis Treatment: Hypothermia
Concurrent with investigations focusing on the pathophysiology of sepsis related laminitis, more clinically oriented studies were being conducted to determine the best prevention or treatment for the devastating lamellar damage observed in the clinical patient. Study subjects in which profound lamellar inflammation was induced
17 using the BWE model exhibited no decrease in inflammation with the administration of
NSAIDS, indicating that although they may help improve an affected animal’s comfort, NSAIDS are unable to significantly alter the progression of the disease.
Similarly, even with the demonstration of an increase in matrix metalloproteinase expression in SRL lamellar tissue, 80-82, treatment of SRL study subjects with marimastat (a broad-spectrum MMP inhibitor) had no protective effect on the lamellar tissue (Underwood PhD).
The first group to test the safety and efficacy of therapeutic hypothermia in the equine digit was Pollitt et al out of the University of Queensland. In their initial study,
Pollitt’s group determined that reducing the hoof wall temperature to less than 10°C for
48 hours by submerging the distal limb (to the level of the carpus) in an ice/water slurry was well tolerated by study subjects, with only one incident of transient distal limb edema which resolved within 24 hours83. This was closely followed by the application of therapeutic hypothermia to laminitis induced using the OF model, in which it was determined that hypothermia markedly reduced the severity of laminitis
(evaluated histologically) and that the protective effects of controlled digital hypothermia (CDH) remained after discontinuation of therapy84-86.
It is important to note that in both of these studies, the hypothermia was initiated at the time of oligofructose administration (immediate hypothermia). In an effort to more closely approximate the presentation of the clinical patient (usually already showing signs of lameness), a more recent study delayed initiation of hypothermia until 12 hours after the administration of the oligofructose bolus (delayed hypothermia). Even with the delayed application of hypothermia, the histological
18 analysis of lamellar tissue from the animals treated with hypothermia revealed a profound protection of lamellar tissue87. This is supported by results of a recent investigation into the clinical use of therapeutic hypothermia for laminitis prophylaxis, in which application of therapeutic hypothermia to colitis patients resulted in a 10% incidence of laminitis, in stark contrast to the 33% incidence of laminitis in the colitis patients not treated with digital hypothermia88.
A relationship between the inflammation seen in the lamellar tissue of SRL patients and the protection conferred by hypothermia was established when the immediate hypothermia model was applied to study subjects in which laminitis was induced using an oligofructose bolus. Lamellar tissue from these subjects (harvested at
OG1 lameness and 24 hours after OF bolus) demonstrated a robust inflammatory response in lamellar tissue from limbs maintained at ambient temperature and a profound decrease in gene expression of inflammatory mediators in the limb treated with therapeutic hypothermia89. These data strongly suggest that not only is the damage observed in the lamellar tissue of SRL study subjects influenced by inflammatory signaling events, but that the protection conferred by digital hypothermia is associated with inhibition of these inflammatory signaling pathways.
Since most clinical cases of SRL are detected after the initial septic insult, it was important to assess the effect of digital hypothermia on lamellar inflammatory signaling in a model in which digital hypothermia was not initiated until a set time point after OF administration (delayed hypothermia). Furthermore, the similarities between the cytoskeletal dysregulation, loss of hemidesmosomal integrity, and stretching on the secondary epidermal lamellae seen in the EHC model of EMSAL and
19 CHO models of SRL made the growth factor signaling pathways shown to be upregulated in the EHC model a logical potential pathway resulting in the lamellar damage seen in OF model of SRL.
20
Chapter 2: Effect of Delayed Digital Hypothermia on Lamellar Inflammatory
Signaling in the Oligofructose Laminitis Model
2.1 Introduction
Recent studies specifically investigating sepsis related laminitis (SRL) have classified the lamellae as a “target organ” of sepsis in the horse, triggering investigation of inflammatory signaling in the early stages of the disease, preceding structural failure of the lamellae 14,90,91. In study subjects in which laminitis was induced using various models of sepsis-related laminitis, a marked increase of a wide range of pro- inflammatory mediators was detected when compared to non-septic control horses
49,63,72,74,89. Similar to the reports from the use of moderate regional hypothermia
(approximately 30°C) in human patients to limit damage to vital organs in cases of inflammatory injury, it has been established that continuous profound digital hypothermia (5-10°C) limits the structural failure of the lamellae both in clinical equine patients with sepsis and experimentally induced laminitis 86,88,89,92,93. Initiation of cryotherapy at the time of oligofructose administration (prior to onset of sepsis or lameness) has been linked to a dramatic decrease of expression of molecules associated with inflammatory signaling when compared to limbs maintained at ambient temperature 89. In a recent study which more closely simulates the time point at which hypothermia is initiated in many clinical equine patients, investigators did not initiate digital hypothermia until the study subjects exhibited lameness (Obel Grade 2) 12,87. 21 The first publication from this study established that, whereas the lamellae from the untreated limb had severe histologic changes consistent with Obel grade 3-4 13, there was minimal histologic evidence of lamellar injury in the limb treated with hypothermia 87. This work confirms that digital hypothermia still has markedly protective effects even when initiated after the onset of lameness; however, the mechanism of this lamellar protection at the later time point has yet to be elucidated.
The aim of the present study was to determine if initiation of hypothermia at the onset of lameness results in a similar inhibition of inflammatory signaling as described when hypothermia was initiated prior to the onset of either sepsis or lameness 89.
Lamellar samples from the same study demonstrating the protective effect of hypothermia initiated at the onset of lameness were used to determine gene expression of pro-inflammatory cytokines, chemokines, endothelial cell adhesion molecules, and
COX-2 87. Due to finding a minimal effect of hypothermia on inflammatory signaling in the present study, lamellar mRNA concentrations from the current study were compared to those of previous SRL studies in which lamellae were harvested at earlier time points to determine if the lack of an “anti-inflammatory” effect of hypothermia in the present study was due to a lack of hypothermia-induced inhibition of inflammatory mediators or simply due to a generalized decrease in the gene expression of inflammatory mediators in late stage laminitis 74,89,91.
2.2 Materials and Methods
Laminitis Induction and Sample Acquisition
22 As described in a previous publication using the same study subjects 87, 8
Standardbred geldings were given an oligofructose bolus (10 g/kg, maximum 4.2 kg) by nasogastric intubation, confined to stocks, and carefully monitored for the onset of lameness using gait evaluation, performed every 4 hours by 2 investigators and using pedometer counts. Pedometer counts were obtained to determine the amount of shifting back and forth between the forelimbs of the study subjects, an early sign of
Obel Grade 1 laminitis. Lameness of 1 or both forelimbs at the walk (Obel Grade 2 laminitis) was the time point used for initiation of digital hypothermia in 1 forelimb
(other forelimb maintained at ambient temperature) and institution of pain management using perineural anesthesia in both forelimbs. Hoof wall surface temperature was measured using hoof wall thermistors attached to data-logging devices as described previously 85. At 36 hours after the development of OG2 laminitis (likely OG3 laminitis due to histologic changes observed)13, horses were euthanized and lamellar tissue samples were collected both from limbs that experienced digital hypothermia and limbs maintained at ambient temperature, as described previously87. Tissue intended for gene expression analysis was immediately snap frozen in liquid nitrogen.
Two other sets of lamellar tissue were analyzed: archived non-septic control
(CON) lamellar tissue from a previous study in which the subjects were not treated with oligofructose 74 and lamellar tissue samples from a previous study in which the tissue samples were harvested at Obel Grade 1 laminitis (OG1) after induction using the oligofructose model 89. In each of these previous studies, the lamellar tissue was harvested in a manner identical to the present study, and the oligofructose laminitis
23 induction model (10 g/kg BW) was identical between the OG1 study and the present study.
RNA Isolation and cDNA synthesis
Total RNA was isolated from lamellar tissue samples using a commercially available preparationa and genomic DNA contamination was prevented by use of
DNase treatment. mRNA then was isolated using a separate commercially available preparationb and cDNA was generated for each tissue sample using reverse transcriptase; cDNA then was frozen at -20°C until RT-qPCR could be performed.
Real Time qPCR
A thermocyclerc was used to perform real-time qPCR, as previously described
89. To determine the effects of cryotherapy on gene expression of inflammatory mediators in lamellar tissue from the current study, previously optimized equine specific primers for E-selectin, intracellular adhesion molecule-1 (ICAM-1), CXCL-1,
CXCL-8 (IL-8), IL-6, IL-1β, CXCL-6, IL-10, MCP-1, MCP-2, COX-2, and 3 housekeeping genes (β-Actin, β2-microglobin, and glyceraldehyde-3-phosphate dehydrogenase) were used to amplify cDNA as described previously 89. The specificity of each primer for the cDNA fragment of interest was confirmed in a previous study using gel electrophoresis 89, melt curve analysis, and sequencing of produced cDNA fragments (Supporting Information 3). Standard curves for each RT-qPCR reaction were generated using previously designed templates 89, and water was used as a negative control. All PCR reactions were performed in 20 µl volumes consisting of 5
µl of sample cDNA and 15 µl of PCR master mix. The master mix included Taq polymerased, uracil-N-glycosylasee, SYBR Green stock solutiond, forward and reverse
24 primers, PCR Nucleotide pluse and PCR buffer prepared on site (20 mmol/L Tris-HCL,
0.05% Tween 20, and a nonionic detergent). Amplification occurred as previously described for 40-45 cycles; the annealing temperature was 1-5°C below melting temperature; extension temperature was set at 72°C for 5 s and fluorescence acquisition for 10 s in the SYBR Green format. Melting curves for each PCR product were acquired by an increase in temperature from 65 to 95°C. Each sample was run in duplicate. To determine if the time of lamellar tissue harvest affected the inflammatory profile in limbs maintained at ambient temperature, RT-qPCR was conducted (as described above) on cDNA obtained from lamellar tissue samples from the present study (OG3), from non-septic archived control lamellar tissue 74, and from lamellar tissue harvested at OG1 89 (both from previous studies). These RT-qPCR reactions were run in identical fashion to those described above, with the exception that, because of limited quantity of cDNA from previous studies, these samples were not analyzed for mRNA concentrations of CXCL-6, IL-10, MCP-1, or MCP-2. Copy number data from genes of interest were normalized using geNorm94software to analyze copy number data of the 3 housekeeping genes to determine which 2 genes received the best acceptable score and then using the data from those 2 genes to make a normalization factor for each sample 89. The amplification data obtained by RT-PCR for the respective genes of interest were divided by the normalization factor of the selected housekeeping genes in the same sample, creating a normalized copy number value. The fold changes for each inflammatory mediator of interest were determined by comparing the copy numbers of the hypothermic (CRYO) limb with the ambient control (NON-
CRYO) limb from the same horse. For the analysis of the inflammatory profile of the
25 ambient limbs, fold changes were determined by comparing the copy number from the
OG1 89 and OG3 limbs (respectively) to the non-septic archived control (CON) 74.
Histological Evaluation
Histological evaluation was performed on formalin-fixed sections (stained by hematoxylin and eosin and periodic acid-Schiff) by 2 blinded observers. A 0-4 grading scale was used as previously described and a grade of 0 was assigned to normal lamellar tissue and a grade of 4 was assigned to sections in which there was complete physical separation of lamellar epidermis from dermis.13,15
Data Analysis
To determine the effects of cryotherapy on the inflammatory profile of laminitic tissue, normalized copy numbers for each inflammatory mediator in CRYO vs NON-CRYO limbs from the current study (OG3) were compared using Wilcoxon signed rank tests.
To compare the mRNA concentrations of inflammatory proteins in ambient limbs from the current study (OG3) with those from non-septic archived control (CON) samples and samples harvested at OG1, RT-qPCR results were compared to each other, respectively, using Mann-Whitney tests. Histopathology scores from the previous publication using the same samples were compared to RT-qPCR results from the lamellar tissue from both CRYO and NON-CRYO limb using Spearman/Pearson correlation tests. All statistics were performed using Graphpad Prismf. Significance was set at P<0.05.
2.3 Results All study subjects developed OG2 lameness between 17 and 21 hours after the
OF bolus dose, as previously reported 87. Hoof wall surface temperature was found to
26 be consistently lower (p< 0.05) in CRYO feet (median 7.1°C) than in NON-CRYO feet
(median 30.2°C), as anticipated.
Effect of digital hypothermia on mRNA concentrations of lamellar inflammatory proteins
Digital hypothermia only resulted in a statistically significant (p<0.05) change in lamellar mRNA concentrations of 2 of the assessed molecules (E-selectin and
ICAM-1), both of which were increased in the hypothermic (CRYO) limb (Table 1,
Figure 4). No significant changes (p<0.05) were noted between the CRYO vs NON-
CRYO limb for proinflammatory cytokines, chemokines, or COX-2 (Table 1).
27
Table 1: Lamellar tissue inflammatory mediator copy number expression data: Forelimbs which were treated with hypothermia (CRYO) compared with untreated (NON-CRYO) limbs Inflammatory Mediator NON-CRYO CRYO E-selectin Fold Increase/NON-RX 3.47 Copy Number 1.70E+4 (7.90E+3-3.53E+4) 5.90E+4 (3.45E+4-1.03E+5) ↑ ICAM-1 Fold Increase/NON-RX 28.25 Copy Number 1.01E+4 (5.00E+3-1.53E+4) 2.85E+5 (3.38E+4-7.04E+5)↑ CXCL-1 Fold Increase/NON-RX 1.50 Copy Number 3.15E+4 (8.91E+3-1.10E+5) 4.74E+4 (2.37E+4-9.55E+4) CXCL-8 (IL-8) Fold Increase/NON-RX 0.51 Copy Number 1.74E+4 (3.96E+3-8.96E+4) 8.92E+3 (2.71E+3-1.42E+4) IL-6 Fold Increase/NON-RX 0.37 Copy Number 3.22E+4 (7.02E+3-8.70E+4) 1.17E+4 (9.55E+2-14.7E+4) IL-1β Fold Increase/NON-RX 0.49 Copy Number 5.86E+4 (1.62E+4-1.20E+5) 2.88E+4 (1.28E+4-1.09E+5) CXCL-6 Fold Increase/NON-RX 0.88 Copy Number 7.79E+3 (4.52E+3-2.61E+4) 6.83E+3 (2.22E+3-1.51E+4) IL-10 Fold Increase/NON-RX 0.96 Copy Number 7.79E+2 (2.89E+2-1.20E+3) 7.49E+2 (4.79E+2-1.61E+3) MCP-1 Fold Increase/NON-RX 2.79 Copy Number 4.32E+3 (1.62E+3-6.45E+3) 1.20E+4 (2.33E+3-2.70E+4) MCP-2 Fold Increase/NON-RX 1.47 Copy Number 1.56E+5 (1.00E+5-3.48E+5) 2.35E+5 (1.46E+5-5.45E+5) COX-2 Fold Increase/NON-RX 0.17 Copy Number 2.30E+4 (4.58E+3-6.75E+4) 3.90E+3 (1.41E+3-3.12E+4)
Lamellar tissue inflammatory mediatory copy number expression data: Forelimbs which were treated with hypothermia (CRYO) compared with untreated (NON- CRYO) limbs. The copy number data is expressed as cDNA copies per normalization factor (interquartile range) in both CRYO and NON-CRYO columns; median fold increase of CRYO copy number (compared to NON-CRYO) is also presented (top number for each mediator) in the CRYO column. ↑ denotes a statistically significant increase in fold change when compared to the untreated limb.
28 Figure 4: Lamellar mRNA concentrations of inflammatory molecules: Limbs undergoing digital hypothermia (CRYO; n=8) vs limbs kept at ambient temperature (NON-CRYO; n=8). Concentrations are expressed as fold changes over CRYO. * denotes a significant difference (p<0.05) between CRYO and NON-CRYO.
29
Table 2: Statistical correlation of lamellar mRNA concentrations of specific mediators with the histological scores (from previous publication) of both hypothermic (CRYO) and untreated (NON- CRYO) limbs. Inflammatory Mediator CRYO NON-CRYO E-selectin Spearman/Pearson R value -0.43 -0.10
p-value ns ns ICAM-1 Spearman/Pearson R value -0.75* 0.44 P-value 0.04 ns CXCL-1 Spearman/Pearson R value -0.58 0.57 P-value ns ns CXCL-8 (IL-8) Spearman/Pearson R value -0.70 0.75* P-value ns 0.04 IL-6 Spearman/Pearson R value -0.68 0.45 P-value ns ns IL-1β Spearman/Pearson R value -0.80* 0.91* P-value 0.02 0.002 CXCL-6 Spearman/Pearson R value -0.36 0.86* P-value ns 0.01 IL-10 Spearman/Pearson R value -0.60 0.54 P-value ns ns MCP-1 Spearman/Pearson R value -0.73* 0.49 P-value 0.04 ns MCP-2 Spearman/Pearson R value -0.55 0.48 P-value ns ns COX-2 Spearman/Pearson R value -0.44 -0.44* P-value ns 0.002
Statistical correlation of lamellar mRNA concentrations of specific mediators with the histological scores (from previous publication) of both hypothermic (CRYO) and untreated (NON-RX) limbs. Ns indicates that the value was not statistically significant. * denotes a statistically significant correlation between the lamellar mRNA concentrations and histological score of the lamina. A negative value indicates an inverse correlation.
30
Correlation of lamellar mRNA concentrations with histological scores
Correlation analysis of lamellar mRNA concentrations and histological scores of the same subjects/digits from a previous study 87 indicated that lamellar mRNA concentrations of ICAM-1, IL-1β, and MCP-1 were inversely correlated (R>0.5, <-
0.5; p<0.05) with the histological scores in CRYO limbs (i.e. higher lamellar mRNA concentrations were seen when the lamellae were intact). In NON-CRYO limbs, the lamellar mRNA concentrations of COX-2, CXCL-6, IL1-β, and IL-8 were positively correlated (R>0.5, <-0.4; p<0.05) with the histological scores (i.e. higher lamellar mRNA concentrations were seen when the lamellae were more separated; Table 2).
Comparison of lamellar mRNA concentrations of inflammatory molecules between non-treated/ambient limbs harvested at OG3 (NON-CRYO OG3), OG1 (NON-CRYO
OG1) and non-septic control animals
Lamellar mRNA concentrations of the majority of inflammatory molecules, with the exception of IL-1β, were increased (p<0.05) in NON-CRYO limbs from the current study when compared with archived lamellar samples from control animals that were not treated with oligofructose (Figure 5, Table 3). When lamellar mRNA concentrations in NON-CRYO limbs of horses in the current study (OG3) were compared with mRNA concentrations in NON-CRYO limbs of horses in which tissue was harvested at onset of OG1 lameness 89, lamellar concentrations of CXCL-1, E- selectin, and ICAM-1 were lower (p<0.05) in the OG3 samples (Table 3).
31
Figure 5: Lamellar mRNA concentrations of inflammatory molecules: Archived non- septic controls (CON; n=6) vs OG3 (harvested 36 hours after the onset of lameness; n=8) vs OG1 (untreated harvested at the onset of OG1 lameness; n=7). The copy number data are expressed as cDNA copies per normalization factor (interquartile range); median fold increase over CON. *denotes significant difference between OG1 and CON (P<0.05); ^denotes significant difference between OG3 and CON.
32 Table 3: Lamellar tissue inflammatory mediator copy number expression data: control (CON) compared with untreated (NON-CRYO; kept at ambient temperature) limbs in subjects in which tissue was harvested at OG1 and 36 hours after onset of OG1 (present study) Inflammatory Mediator CON OG1 NON- 36 h after OG1 CRYO NON-CRYO E-selectin Fold Increase/CON 14.29* 2.82*^ Copy Number 4.07E+3 (2.78E+3- 5.80E+3 (2.08E+4- 1.14E+4 (5.25E+3- 8.43E+3) 6.62E+4) 3.38E+4) ICAM-1 Fold Increase/CON 180.49* 1.62^ Copy Number 5.74E+3 (2.12E+3- 1.04E+5 (2.26E+5- 9.38E+3 (3.31E+3- 9.29E+3) 4.61E+6) 1.46E+3) CXCL-1 Fold Increase/CON 285.72* 18.53*^ Copy Number 7.43E+2 (5.97E+2- 2.12E+5 (1.74E+5- 1.38E+4 (3.46E+4- 1.23E+2) 3.91E+6) 2.49E+4) CXCL-8 (IL-8) Fold Increase/CON 65.13* 46.48* Copy Number 4.17E+2 (3.18E+2- 2.72E+4 (1.96E+4- 1.94E+4 (5.48E+3- 5.05E+2) 1.05E+5) 1.02E+5) IL-6 Fold Increase/CON 258.81* 91.78* Copy Number 2.57E+2 (8.70E+2- 6.66E+4 (3.84E+3- 2.36E+4 (6.12E+3- 5.22E+2) 1.47E+5) 5.04E+4) IL-1β Fold Increase/CON 4.46* 3.87 Copy Number 6.03E+3 (4.02E+3- 2.69E+4 (1.85E+4- 2.34E+4 (4.74E+3- 9.28E+3) 5.52E+4) 5.08E+4) COX-2 Fold Increase/CON 58.11* 10.19* Copy Number 9.21E+2 (4.64E+2- 5.34E+4 (1.42E+3- 9.35E+3 (2.88E+3- 2.14E+3) 1.73E+5) 5.67E+4)
Lamellar tissue inflammatory mediator copy number expression data: control (CON) compared with untreated (NON-CRYO; kept at ambient temperature) limbs in subjects in which tissue was harvested at OG1 and 36 hours after onset of OG1 (present study). The copy number data are expressed as cDNA copies per normalization factor (interquartile range); median fold increase over CON. *denotes significant difference from CON (P<0.05); ^denotes significant difference in OG1 NON-CRYO compared with 36h after OG1 NON-CRYO
33 2.4 Discussion
We failed to find a decrease in lamellar concentration of molecules associated with inflammatory signaling when digital hypothermia was initiated after the onset of lameness in the OF model of SRL, in contrast to the marked decrease in gene expression of these same molecules observed when hypothermia was initiated at the time of administration of a carbohydrate overload (i.e. before signs of sepsis or lameness) in a previous study 89. Possible explanations for this lack of inflammatory inhibition are: a difference in the severity of lamellar injury between the studies, a difference in inflammatory profile as a consequence of the later time point at which the lamellar tissue was evaluated in the current study (i.e. minimal inflammation may be present to inhibit), or the protective effects of digital hypothermia are not mediated through inflammatory pathways.
The possibility of a lack of induction of lamellar injury being responsible for the minimal effect of hypothermia on inflammatory signaling in the present study is contradicted by the histological evaluation of lamellae from these same animals in a previous report that identified a high degree of lamellar separation consistent with
OG3-4 laminitis in limbs maintained at ambient temperature (NON-CRYO) 87.
Additionally, evidence of marked inflammation in NON-CRYO limbs in the current study is provided by comparison of lamellar mRNA concentrations of inflammatory mediators in NON-CRYO limbs from the current study with mRNA concentrations of inflammatory mediators in archived lamellar tissue from non-septic control animals
74,89. These data indicate that NON-CRYO limbs in the current study exhibited up to
90-fold increases (p<0.05) in lamellar mRNA concentrations of several classes of
34 inflammatory molecules (E-selectin, CXCL-1, IL-8, IL-6, and Cox-2) when compared to the low mRNA concentrations of these same molecules in non-septic control samples. The increase in gene expression of inflammatory mediators in these limbs, coupled with the histological evidence of lamellar separation in these samples, confirms that both severe lamellar injury and increased inflammatory signaling were present in the untreated limbs from our study.
Although prominent inflammatory signaling still was present in the current study when compared to non-septic controls, further evaluation of the inflammatory gene expression data indicates that the severity of lamellar inflammation in the non- treated limbs (NON-CRYO, OG3) in the current study were not as marked as reported in tissue samples harvested from non-treated (NON-CRYO) limbs at an earlier stage of laminitis (OG1) in a previous study 89. The significantly decreased gene expression of E-selectin, ICAM-1, and CXCL-1 in samples of NON-CRYO limbs from the current study (OG3) when compared with the previous study (OG1) indicates a lower degree of lamellar inflammatory signaling at OG3 in the OF model of sepsis-related laminitis.
This finding is consistent with recent work performed on the OG3 time point in the traditional (starch gruel/wood flour) model of laminitis (Belknap and Eades laboratories, unpublished data), in which lower lamellar concentrations of inflammatory mediators were present than previously reported for the OG1 time point with the same model 89. Although continuous perineural analgesia used in our study subjects precluded assignment of laminitis grades beyond OG2, the degree of lamellar separation evident on histological evaluation of the samples is indicative of OG3 laminitis 13. Interestingly, perineural analgesia itself has been reported to cause a
35 decrease in gene expression of inflammatory proteins, and a relationship between cytokines and neuro-hormonal feedback and perineural anesthesia and suppression of inflammation has been observed in both humans and animals 95-98.Although this relationship has yet to be investigated in laminitis, a recent study of inflammatory signaling in sepsis-related laminitis was conducted without the use of perineural anesthesia. This study demonstrated a similar waning of lamellar inflammatory signaling at a later time point, which indicated that lamellar inflammation waned at later time points compared to early/OG1 laminitis with or without perineural anesthesia
(Belknap and van Eps, unpublished data).
The most notable finding from the present study is that, although multiple inflammatory mediators still were markedly increased in untreated limbs when compared to non-septic controls, digital hypothermia did not cause a significant decrease in the lamellar mRNA concentrations of any of the cytokines, chemokines, or endothelial adhesion molecules associated with inflammatory signaling. Interestingly,
ICAM-1 and E-selectin, 2 adhesion molecules important in leukocyte emigration, were the only mediators in the present study to be significantly different in CRYO versus
NON-CRYO limbs. However, in contrast to the previous study in which hypothermia initiated earlier induced a significant decrease in lamellar concentrations of these molecules, mRNA concentrations of both were increased in the current study.
Although there is a paucity of data in the human literature about this increase in endothelial adhesion molecules under the influence of hypothermia, the marked degree of hypothermia reached in our study (7°C) as compared to the moderate hypothermia
(30°C) described in the human literature could be responsible for the lack of similar
36 findings. The apparent disparity between the effect of hypothermia on inflammatory gene expression and the protection against lamellar injury provided by digital hypothermia in the present study is further highlighted by the correlative statistical analysis of the fold changes and histological scores from lamellar samples. As seen in
Table 2, lamellar mRNA concentrations of the inflammatory mediators ICAM-1, IL-
1β, and MCP-1 were inversely correlated with histology scores from the same samples, indicating that the highest mRNA concentrations of these inflammatory mediators were obtained from lamellae that had the least histologic signs of injury and separation in the hypothermic limbs. This discrepancy between hypothermic protection and inflammation is not unprecedented in therapeutic hypothermia in other species, which has led to the investigation of other possible links between hypothermia and tissue protection including the effect of hypothermia on energy metabolism, mitochondrial dysfunction, free radical production, and cold shock proteins 99.
The lack of inhibition of inflammatory mediators in lamellar tissue samples in the current study, when evaluated in conjunction with the degree of lamellar protection provided by cryotherapy in the same study subject, indicates that the protective effects of digital hypothermia may not be mediated through inflammatory pathways. This finding correlates well with investigations in the human literature in which, although mild hypothermia is the standard of care in the intensive care units of many hospitals due to its protective effect from tissue damage secondary to traumatic brain injury, spinal cord injury, cardiac arrest, and neonatal hypoxic encephalopathy, the specific mechanism of protection has not been elucidated and appears to be more than inhibition of inflammatory signaling 100-102. Thus, additional work investigating
37 alternate pathways as the protective mechanism of action of hypothermia in sepsis- related laminitis is warranted, with the ultimate goal being the discovery of a specific and targeted pharmaceutical intervention that would prevent the development and progression of laminitis in horses with sepsis.
38
Chapter 3: Controlled Digital Hypothermia in Sepsis Related Laminitis at
Clinically Relevant Timepoints
3.1 Introduction
Sepsis-related laminitis (SRL) is a common sequela to diseases associated with systemic sepsis in the horse, commonly resulting in extensive lamellar injury and subsequent displacement of the distal phalanx within the hoof capsule. Multiple reports regarding the pathophysiology of sepsis-related laminitis, primarily studied in the black walnut extract model or carbohydrate overload models in which an intragastric bolus of either corn starch gruel or oligofructose is administered,43,44,47 have supported a central role of inflammation in the form of lamellar leukocyte influx and markedly increased lamellar concentrations of inflammatory mediators.49,63,73,74,103-
110 In regards to therapy, SRL is the only type of laminitis in which an effective therapy, controlled digital hypothermia (CDH, also termed “cryotherapy”), has been established. Using the oligofructose model of SRL, CDH has been reported to effectively protect lamellae from structural failure when initiated prior to the onset of systemic sepsis (initiated at the time the OF bolus was administered)86 and when initiated at the onset of Obel Grade 2 (OG2) laminitis.87 In a retrospective clinical study in which CDH was administered prophylactically to equine patients exhibiting signs of enterocolitis, it was found to significantly decrease the incidence of SRL.88
Determining the mechanism by which CDH protects the lamellae is important, as it
39 may provide therapeutic targets for pharmaceutical drugs to supplement or replace the often cumbersome technique of CDH.
Due to a previous study demonstrating profound inhibition of a wide array of inflammatory mediators when CDH was instituted prior to either systemic sepsis or lamellar disease,89 CDH has been purported to limit lamellar injury due to its anti- inflammatory effects. However, a more recent study in which CDH was initiated at the onset of clinical lameness (OG2) did not find the same degree of inhibition of lamellar inflammation despite still conferring a dramatic protective effect upon the lamellae.87,111 Similarly, a recent study investigating the effects of therapeutic hypothermia on leukocyte infiltration in the OF SRL model found a decrease in leukocyte (MAC387(+)) counts at only one time point, indicating that leukocyte infiltration may have less of a role in lamellar tissue damage than previously thought112.
The current study was performed to assess the effect of CDH on lamellar inflammatory signaling and leukocyte infiltration when CDH was instituted at the time point in which it is commonly instituted in the clinical situation, the onset of clinical signs of systemic disease. In conrast to previous studies of CDH, a non-septic control group was incorporated into the study to both 1) assess the effect of CDH on lamellar signaling in the normal limb, and 2) to be able to accurately assess whether CDH in septic limbs effectively inhibit inflammatory signaling to the level observed in the normal limb. Additionally, in contrast to the study in which minimal anti- inflammatory effect was detected when CDH was initiated at the onset of OG2 laminitis, 87,111 samples were harvested at an earlier time point (prior to the onset of
40 severe lamellar separation/failure) to avoid the effect that extensive structural tissue injury may have on inflammatory signaling. The results of the present study support the premise that lamellar protection provided by CDH is not due to a broad inhibition of inflammatory signaling.
3.2 Materials and Methods:
This project was approved by the University of Queensland Animal Ethics Committee
(AEC) that monitors compliance with the Animal Welfare Act (2001) and The Code of
Practice for the care and use of animals for scientific purposes (current edition). All animals were monitored continuously by the investigators.
Laminitis Induction, Controlled Digital Hypothermia, and Sample Acquisition
Fifteen Standardbred geldings (aged 3-11 years) with no evidence of lameness or gross abnormalities of the feet were randomly assigned to one of two groups: a control group
(CON; n=8) not administered oligofructose (OF), and an OF group (n=7), in which nasogastric intubation of a 10g/kg bolus of oligofructose, up to a maximum dose of 4.2 kg laminitis was administered. Although the oligofructose (OF) group initially contained 8 animals, one study subject was excluded for behavioral reasons. Each study subject was confined to stocks for the duration of the experiment, having constant access to hay and water.
Hoof wall temperature was recorded in all study subjects using thermistors attached to the dorsal hoof wall of both forelimbs, and human pedometer devices were attached to the forearm of both forelimbs to record the frequency of weight shifting. Study subjects were monitored carefully for onset of lameness using subjective gait evaluation.
Analgesia using a combination of continuous peripheral nerve block (CPNB) as
41 previously described113 and phenylbutazone (4.4mg/kg) was initiated in any study subjects exhibiting signs of lameness.
Controlled digital hypothermia
In order to replicate the time point at which CDH is commonly instituted in clinical cases, 12 hours post OF administration was chosen due to the fact that, in two previous studies using the same OF protocol,44,89 horses exhibited systemic signs of disease
(e.g. diarrhea) at 10-14 hours post OF administration. Thus, 12 hours after either the
OF bolus (OF group) or the beginning of the confinement in stocks (CON group), controlled digital hypothermia was initiated in one randomly assigned forelimb of each study subject (ICE or AMB). Controlled digital hypothermia was achieved by placing the ICE limb in a rubber boot (Bigfoot Ice boot) which contained a 50% water and
50% ice mixture to the level of the proximal metacarpus. The cubed ice level in the rubber boots was constantly maintained over the course of the experiment.
Sample Acquisition
Due to the concern that the severe physical destruction of the lamellae from ambient limbs in the delayed CDH study (in which CDH was instituted at the onset of lameness) may have influenced inflammatory signaling and confounded the comparison of inflammatory signaling with the relatively intact lamellae in the hypothermic limb87, lamellae were harvested at an earlier time point in the current study. Thus, at 36hours after the beginning of the experimental period (i.e. 36 h post
OF administration in the OF group), each study subject was humanely euthanized with pentobarbital sodium (20mg/kg bwt IV). The dorsal lamellae were dissected from the hoof and third phalanx in each forelimb (AMB and ICE) from each study subject. The
42 sections were either snap frozen (for RT-qPCR) or fixed in formalin and processed for light microscopy.
Histological Evaluation
H&E and PAS were used on formalin fixed sections, which were randomized and coded for histological analysis by 2 blinded observers (AE and CP). Histopathological scores were assigned using a system previously described by Pollit7: a 0-4 scale with a score of 0 assigned to normal dermal and epidermal interdigitation and a score of 4 indicating no association between dermal and epidermal tissue on the section.
RNA isolation, cDNA synthesis, and Real Time qPCR
Flash frozen lamellar tissue samples were processed using a commercially available preparation (Absolutely RNA Miniprep kit, Agilent Technologies, Stratagene Products
Division, La Jolla CA) to isolate total RNA. mRNA was then isolated using a separate preparation (mRNA extraction kit, Roche Applied Science, Indianapolis IN) and reverse transcriptase was used to generate cDNA for each tissue sample. This cDNA was then frozen at -20°C until RT-qPCR could be conducted.
Real-time qPCR was performed using a thermocycler (Roche 480®; Roche Applied
Science, Indianapolis IN), as previously described. Equine specific primers (specificity confirmed in a previous study) for COX-1, COX-2, CXCL-1, CXCL-6, E-selectin,
ICAM-1, IL-1β, IL-6, IL-8, IL-10, MCP-1, MCP-2, and 3 housekeeping genes were used to amplify cDNA as previously described7,89. Previously designed templates were used to generate standard curves for each RT-qPCR reaction; distilled water was used as a negative control and each sample was run in duplicate. Amplification was performed in a 20uL reaction volume consisting of 15uL of PCR master mix (made on
43 site) and 5uL of sample cDNA. The master mix included Taq polymerase, SYBR
Green stock solution, uracil-N-glycosylase, forward and reverse primers, PCR
Nucleotide plus, and PCR buffer (20mmol/L Tris-HCl, 0.05% Tween20, and a nonionic detergent). The thermal cycle was programmed as previously described with an initial denaturation period followed by 40-45 cycles of amplification: the annealing temp was 1-5°C below melting temperature; 72° for 5s for extension; and 10s for acquisition. An increase in temperature from 65-95°C was used to acquire melting curves for each PCR product. Copy number data were normalized using GeNorm software and 3 housekeeping genes (β-Actin, β2-microglobin, and glyceraldehyde-3- phosphate dehydrogenase). Two housekeeping genes were used to make a normalization factor for each sample, and the amplification data produced by RT-qPCR were divided by the normalization factor, creating a normalized copy number value.
The normalized copy numbers of the hypothermic control limb (ICE-CON), laminitic control limb (AMB-OF), and hypothermic laminitic limb (ICE-OF) were compared to the copy numbers of the ambient control limb (AMB-CON) to determine a fold change for each inflammatory mediator of interest.
Leukocyte Assessment
Paraffin embedded, formalin-fixed tissues were sectioned to 4-µm thickness and stained for MAC387/calprotectin (Abcam Cambridge, MA, USA) and CD163 (Cosmo
Bio Carlsbad, CA, USA). Immunohistochemistry detection was conducted using the avidin-biotin complex method. For detection of MAC387 cells, each section was deparaffinized, treated with protease solution (Proteinase-K (Fisher Scientific
Pittsburg, PA, USA), with concurrent quenching of endogenous peroxidase activity
44 using 3% hydrogen peroxide. The slides were then incubated for 1h at 22°C using a blocking solution (2% serum) and subsequently incubated at 4°C overnight using mouse monoclonal anti-human MAC387 antibody. The slides were then incubated with biotinylated secondary antibody (Vector Laboratories Burlingame, CA, USA) and then with avidin-horseradish peroxidase complex (Vector Laboratories Burlingame,
CA, USA). After peroxidase incubation, DAB chromagen was used to develop the signal, and tissues were counterstained with Hematoxylin.
Staining for CD163 positive cells was carried out as above, with the exception that a primary antibody for CD163 was utilized.
An automated scanning robot was used to randomly acquire 5 whole-slide digital images from each slide (40XHPF). An operator blinded to the origin of the tissue manually counted MAC387-positive and CD163-positive leukocytes in each individual image.
Data Analysis
The distribution of RT-qPCR data was found to be skewed to the right and was therefore transformed logarithmically after which it was distributed in a fashion more closely approximating Gaussian distribution. A linear mixed model for longitudinal data was used to analyze the data. The model was specified with logarithmically transformed mRNA copy number data as the dependent variable and treatment (ICE or
AMB), induction (OF or CON), and interaction between treatment and induction as fixed effects. Horse ID was specified as a random effect. Regression statistical analyses were performed using JMP statistical analysis software.
45 The number of lamellar MAC387(+) and CD163(+) cells was compared between ICE and AMB horses in both OF and CON groups. Graphical analysis of the data revealed non-Gaussian distribution, so the data was logarithmically transformed after which it was distributed in a manner more closely approximating Gaussian distribution. The data were then analyzed using a linear mixed model for longitudinal data, with cell counts specified as the dependent variable and treatment (ICE or AMB), induction (OF or CON), and interaction between treatment and induction as fixed effects. Horse ID was specified as a random effect. Regression statistical analysis was performed using
JMP statistical analysis software.
The median histological scores were compared between the treated OF (ICE OF) and untreated OF (AMB OF) lamellar tissue using Wilcoxon signed ranks tests using
Graphpad Prism. Significance was set at p<0.05.
Correlation between histopathology scores and gene expression of inflammatory mediators was determined by comparing RT-qPCR results from the lamellar tissue of both AMB-OF and ICE-OF limbs to histopathology scores from the same tissue using
Spearman/Pearson correlation tests. Correlative statistics were performed using
Graphpad Prism with significance set at P<0.05.
3.3: Results
Induction of sepsis in study subjects
All horses in the OF group developed pyrexia (rectal temperature>38.4°C) within 18h of the oligofructose bolus (median/interquartile range, 14/10-14), with the exception of one study subject who was administered 2.0g phenylbutazone IV at 10h due to lameness. All horses in the OF group developed tachycardia (>45 beats/min) within
46 16h of the oligofructose bolus (median/interquartile range, 16/10-16.5). Pedometer data in OF study subjects revealed a significant increase in limb movement of the ambient limbs (P<0.05) when compared to limbs subjected to controlled digital hypothermia.
Hoof wall surface temperature data obtained during the experiment was 5.91, 1.29°C
(median, interquartile range) for ICE OF limbs, 27.74,2.96°C for the AMB OF limbs,
5.33, 1.35°C for the ICE CON limbs, and 24.49, 5.18°C for the AMB CON limbs.
Effect of digital hypothermia on histopathological scores of lamellar tissue
Median histological scores of the middle lamellar sections were significantly lower in the limbs treated with digital hypothermia (ICE OF) (0 [0,0]), compared with the limbs maintained at ambient temperature (AMB OF) 2{1.5,2.5]).
Effect of digital hypothermia on mRNA concentrations of lamellar inflammatory proteins
The linear mixed model included the higher order term of the interaction between induction and treatment and the main effects of induction and treatment.
Effect of digital hypothermia on mRNA concentrations of lamellar inflammatory cytokines
When considering the main effect of the interaction between induction (OF) and treatment (ICE), induction of SRL via administration of oligofructose bolus did not result in a change in the mRNA levels of any of the inflammatory cytokines, nor did treatment with digital hypothermia (ICE) result in a change in gene expression of the inflammatory cytokines.
47 When the main effect of induction (OF) was considered, alone, increases in gene expression of IL1β and IL6, were observed (p<0.0001).
When the main effect of treatment (ICE) was considered, alone, a decrease in gene expression of IL-6 was discovered in the lamellar tissue from the limbs subject to digital hypothermia (p<0.01).
Effect of digital hypothermia on mRNA concentrations of lamellar inflammatory chemokines
When considering the main effect of the interaction between induction (OF) and treatment (ICE), induction of SRL via administration of oligofructose bolus resulted in an increase in gene expression of CXCL1 and MCP2 (p<0.05). Treatment AND induction resulted in an increase in gene expression of MCP2 which was greater than the increase seen with induction alone (p<0.01).
When the main effect of induction (OF) was considered, alone, increases in gene expression of CXCL1, IL-8/CXCL8, MCP1, and MCP2 were observed (p<0.05).
When the main effect of treatment (ICE) was considered alone, an increase in gene expression of MCP2 and CXCL1 was discovered in the lamellar tissue from the limbs subject to digital hypothermia (p<0.05).
Effect of digital hypothermia on mRNA concentrations of lamellar cycloxygenases and cell adhesion molecules
When considering the main effect of the interaction between induction (OF) and treatment (ICE), induction resulted in an increase in gene expression of COX-2
(p<0.05). In the case of COX-1, induction in the absence of treatment resulted in a decrease in gene expression (p<0.0001). Treatment resulted in a decrease in gene
48 expression of COX-2 (p<0.01). Induction (OF) and treatment (ICE) resulted in an increase in gene expression of ICAM (p=0.03).
When the main effect of induction (OF) was considered, alone, increases in gene expression of COX2, ICAM, and e-Selectin were observed (p<0.05). A decrease in gene expression of COX1 was observed (p<0.0001).
When the main effect of treatment (ICE) was considered, alone, a decrease in gene expression of COX2 was observed (p<0.001). Conversely, an increase in gene expression of COX1 and ICAM was observed in lamellar tissue from limbs subject to digital hypothermia (p<0.005).
Summary of effect of digital hypothermia on mRNA concentrations of lamellar inflammatory proteins
OF induction resulted in increased lamellar concentrations of cytokines (IL1β and
IL6),chemokines (CXCL1, CXCL8, MCP1, and MCP2), COX-2 and cell adhesion molecules(ICAM-1 and E-selectin). The treatment of OF-induced horses with CDH resulted in significant decreases in lamellar mRNA concentrations of IL-6 and COX-2 and increased lamellar mRNA concentrations of ICAM-1 and MCP-2 (Table 4).
49
Inflammatory Mediator
IL1β IL6 IL8 IL10 CXCL1 CXCL6 MCP1 MCP2 COX1 COX2 ICAM e-SEL
Treatment p=0.34 p<0.01 p=0.08 p=0.06 p=0.03 p=0.12 p=0.56 p=0.02 p<0.001 p<0.0001 p=0.0001 p=0.06
ICE 15244.9 3229.2 12926. 1784.7 22697. 2052.9 2090.2 195633.8 879404.3 2460.2 45297.2 20743.7
AMB 12469.0 12657.4 6063.2 3415.2 15244. 3415.2 1758.1 124492.4 324811.4 8299.9 16236.2 10583.0
Regression Regression Applied Model Induction p<0.0001 p<0.0001 p<0.01 p=0.61 p<0.01 p<0.01 p=0.01 p<0.0001 p<0.0001 p<0.0001 p<0.0001 p<0.0001
OF 37571.5 21843.6 58454. 2326.2 52891. 11660.9 11407.2 308044.6 199187.1 19399.5 57354.1 56162.2
5 0
CON 5064.4 191.1 1340.8 2620.2 6535.5 601.8 321.8 79063.0 1435465.9 1052.6 12823.1 3908.9
Interaction p=0.085 p=0.385 p=0.85 p=0.88 p<0.01 p=0.53 p=0.79 p<0.01 p<0.0001 p<0.001 p=0.03 p=0.92
ICE-OF 50261.8 89500.5 88521. 1639.3 85135. 8184.5 11968.1 518658.0 643707.7 5681.7 13052.8 77265.3
ICE-CON 4623.9 116.6 1887.5 1943.0 6051.1 514.9 365.0 73791.6 1202604.3 1064.2 15740.6 5569.16
AMB-OF 28057.2 510936.2 38599. 3301.1 32859. 16597.4 10872.8 182955.8 61635.9 66171.2 25235.3 40863.8
AMB-CON 5541.4 313.2 951.5 3533.3 7058.6 702.7 284 84710.9 1711704.1 1041.1 10446.3 2740.79
Table 4: The LSMeans of the copy number data predicted using each model are presented, along with the F test value (p-value) for each model when applied to each inflammatory mediator.
50
Effect of digital hypothermia on lamellar leukocyte concentration
The linear mixed model included the higher order term of the interaction between induction and treatment and the main effects of induction and treatment.
Effect of digital hypothermia on lamellar concentration of MAC387-positive cells
When considering the main effect of the interaction between induction (OF) and treatment (ICE), induction of SRL via administration of oligofructose bolus resulted in an increase in MAC387(+) cells in lamellar tissue. Although the lamellar tissue from the ICE hoof of study subjects administered oligofructose had an increase in
MAC387(+) cell over the lamellar tissue from study subjects not given OF, the ICE OF lamellar tissue still had significantly less MAC387(+) cells than the lamellar tissue from the hoof left at ambient temperature (p<0.0001) (Figure 6).
Effect of digital hypothermia on lamellar concentration of CD163-positive cells
When considering the main effect of the interaction between induction (OF) and treatment (ICE), the lamellar tissue from the hoof subjectto digital hypothermia in the study subjects administered oligofructose (ICE OF) had the least amount of CD163(+) cells. The lamellar tissue from the ambient hoof in the study subjects administered oligofructose (AMB OF) had the greatest amount of CD163(+) cells. The lamellar tissue from the study subjects that were not administered oligofructose (AMB CON,
ICE CON) had a greater amount of CD163 (+) cells than the ICE OF tissue, but cell counts were less than that of the AMB OF lamellar tissue (p=0.0011) (Figure 6).
51
Figure 6: Assessment of lamellar leukocyte concentration.
3.4: Discussion
This is the first study to determine the effect of hypothermia on normal lamellae. These data demonstrate no significant effect of hypothermia on inflammatory mediator gene expression (at least at the transcriptional level) in normal lamellar tissue across a broad array of inflammatory mediators including cytokines, chemokines, leukocyte adhesion molecules (adhesins), and isoforms of COX. Having these data allows us to distinguish that any effect of hypothermia on lamellae in the OF group is not a general effect of hypothermia on healthy or diseased tissue; rather, the effects of hypothermia on lamellar signaling are distinct effects on inflammatory
52 mediators in lamellar tissue from patients with sepsis related laminitis. Of primary interest in the current study is the lamellar expression of several inflammatory mediators in which hypothermia appeared to exacerbate the response to sepsis. For three chemokines (MCP-2, CXCL-1, IL-8) and the leukocyte adhesion molecule
ICAM-1, sepsis itself did not significantly increase mediator expression, whereas the mixture of sepsis and hypothermia induced a significant increase in mRNA concentration of these mediators in the lamellar tissue. Thus, as leukocyte extravasation into tissue is reported to both 1) be stimulated expression of chemokines and endothelial adhesion molecules, and 2) be a central factor associated with organ injury in human sepsis and lamellar injury in SRL 114,115, the combination of chemokines and leukocyte adhesins induced by local hypothermia in the septic equid would appear to be deleterious to lamellar function and integrity. Interestingly, whereas these results differ greatly from an earlier study in which hypothermia instituted prior to the onset of systemic disease resulted in a profound decrease in most inflammatory mediators assessed including the same leukocyte adhesins, the results are similar to a more recent study where hypothermia instituted at the onset of OG2 lameness (a later time point than the current study) resulted in lamellar protection but also resulted in the same increase in the leukocyte adhesion molecules as noted in the current study.
The increase in MAC387(+) and CD163(+) cells in the tissue from animals in which SRL was induced is consistent with the leukocyte influx previously documented in both BWE, CHO-overload, and OF induced laminitis models42,106,112. The significant decrease in both MAC387(+) and CD163(+) positive cells in septic lamellar tissue
53 treated with therapeutic hypothermia, however, starkly contrasts with a recent investigation into the effects of hypothermia on OF-induced laminitis, in which hypothermia had no effect on leukocyte influx at the developmental time point, and caused a decrease in only MAC387(+) cells in the lamellar tissue at the OG1 timepoint112. The decrease in both MAC387(+) cells and CD163(+) cells observed in the ICE OF tissue in our current study correlates well with investigations in human medicine in which therapeutic hypothermia was found to shift the balance away from
M1(pro-inflammatory, represented by MAC387(+) cells) and toward M2 (anti- inflammatory) microglial phenotypes in a post-traumatic brain injury model116, as well as decreasing microglial activation and migration in vivo117. Recently, the role of the microglial cell (resident macrophage in neuronal tissue), has come under increased focus as a polarizing agent in neuronal inflammation post trauma or hypoxemic insult in human patients, prompting multiple investigations into the effects of therapeutic hypothermia on microglial cells118. Since therapeutic hypothermia has repeatedly been shown to mitigate brain damage in human patients (whether post-shock, cardiac bypass, stroke, or hypoxic ischemic encephalopathy), any correlations that can be drawn between investigations into neuroinflammation in the human patient and damage to lamellar tissue in the equine septic patient warrant further consideration100,102,119,120. Interestingly, despite the widespread use of therapeutic hypothermia in the human critical care field, investigations into the exact mechanism of cryoprotection have yet to be elucidated121-124.Similar to previous studies assessing lamellar concentrations of inflammatory molecules in experimental models of SRL,
49,70,72,74,105,107,108 lamellar concentrations of many inflammatory signaling molecules
54 including cytokines, chemokines, adhesion molecules, and COX-2 were increased in the untreated limbs of animals administered oligofructose in the current study.
However, although digital hypothermia in the current study was found to confer the same structural protection to the lamellae as reported in previous SRL studies, 85,89 this study failed to find the same decrease in a broad array of molecules associated with inflammatory signaling in limbs undergoing digital hypothermia as was reported in a study in which hypothermia was instituted at the same time the administration of the oligfructose bolus was performed.89 In an attempt to more closely approximate the clinical setting, controlled digital hypothermia was not initiated in study subjects until
12h post-OF bolus in the current study (at approximately the time that the animals were previously documented to exhibit diarrhea in this model). 89 Because this is commonly the time point in clinical cases of enterocolitis when an animal is considered to be at risk of SRL and CDH is instituted, 88 the results of this study are likely more clinically relevant than those in which CDH is instituted prior to the administration of a carbohydrate overload. Additionally, the harvesting of lamellar tissue prior to severe structural failure of the lamellae in the current study likely gives a more accurate assessment of lamellar signaling related to sepsis (vs. signaling induced by traumatic injury). In light of these adjustments (the delayed initiation of CDH and earlier tissue harvest) and the inclusion of a control group undergoing CDH, the lamellar tissue samples from this study comprise the most representative sample of the practical implementation of controlled digital hypothermia to treat sepsis related laminitis in the equine patient. As such, the fact that hypothermia, while still providing structural protection to the lamellae, only resulted in significant decreases in lamellar
55 concentrations of IL-6 and COX-2 indicate that either: 1) investigators need to focus on signaling related to these molecules, and/or 2) hypothermia is working through mechanisms unrelated to inflammatory signaling.
Whereas therapies to inhibit COX activity have had conflicting results in the treatment of human sepsis, 125 minimal efficacy was reported when non-selective
NSAIDs were assessed in an equine model of SRL. 126 IL-6-related signaling may merit further attention. The fact that IL-6 signaling has consistently shown the greatest increases in the lamellae in different models of sepsis-related laminitis, 49,74,127 combined with the fact that it has been correlated with organ injury and death in multiple studies of human sepsis,128-130 indicate that signaling induced by this cytokine should be intensively investigated in SRL. IL-6 signaling through the GP130 receptor has recently received a great deal of attention in inflammatory disease states, as it has been reported to be important in the transformation of epithelial cells to tumor cells
(the first stage of which is termed epithelial to mesenchymal transition; EMT) in diseases such as inflammatory bowel disease.26,131,132 Of interest to laminitis, where loss of structural integrity of the lamellae is purported to occur due to disruption of the lamellar epithelial cell cytoskeleton and adhesion of these cells to the underlying matrix,14,18,41,133,134 is the fact that two of the first events to occur in EMT are dysregulation of cellular adhesion and disruption of cytoskeletal dynamics. 135 Thus, although the lamellar epithelial cells obviously do not progress past EMT into the proliferative and metastatic stages of neoplastic cells, similar epithelial signaling driven by IL-6 may lead to the lamellar epithelial cell stretching and separation from the lamellar dermis, the two central histologic findings documented to occur with lamellar
56 failure in both sepsis-related and endocrinopathic laminitis. 42 In addition to further work investigating IL-6 related signaling, investigators need to continue to use other experimental techniques (e.g. next generation sequencing, kinomic arrays) which screen a broad array of signaling pathways both related and unrelated to inflammation in order to find effective therapeutic targets for pharmacologic management of SRL.
57
Chapter 4: Growth Factor Signaling in Sepsis Related Laminitis
4.1 Introduction
Despite billions of dollars of funding being spent on research related to the role of inflammatory signaling in organ injury/dysfunction in human sepsis, there is still no effective therapy for the septic human patients. Although many of the same pathophysiologic events-primarily related to inflammatory signaling-have been documented to occur in lamellar failure in the septic horse, equine research both in the research laboratory and clinical arena has resulted in only one effective prophylactic therapy for sepsis-related laminitis, continuous digital hypothermia (CDH; also known as “cryotherapy”) 90. Recent research has focused on the effect of CDH on the pathophysiologic events documented to occur in SRL, in the hopes of finding a therapeutic target for development of an effective pharmaceutical agent to aid in the treatment of this disease. The most commonly used model for the study of SRL is the oligofructose (OF) model, in which an intragastric bolus of carbohydrate results in clinical signs of sepsis within approximately 12 hours and signs of laminitis within approximately 24 hours. Whereas, an early study in which CDH was instituted at the same time as OF administration resulted indicated a broad inhibition of multiple types of inflammatory signaling, a more recent study in which CDH was instituted 12 h post
OF administration-a time relevant to the clinical situation where CDH is commonly
58 instituted when the animals first exhibit signs of sepsis-resulted only in the inhibition of two inflammatory mediators, IL-6 and COX-289,111.
The two primary central histologic events documented to occur in both sepsis- related laminitis and endocrinopathic laminitis are lamellar stretching and dysadhesion of the lamellar basal epithelial cells from the underlying basmement membrane and dermis11,13,14,27,41. Lamellar stretching has been proposed to occur due to a loss of structural integrity of lamellar epithelial cells due to cytoskeletal dysregulation. These two events likely to play a central role in structural failure of the lamellae, cytoskeletal dysregulation and cellular dysadhesion from the underlying matrix, are two of the initial events occurring when epithelial cells undergo transformation to tumor cells
(i.e., epithelial to mesenchymal transition [EMT])135. Interestingly, IL-6, working through its receptor gp130, has been found to be important in inducing the transformation of epithelial cells into tumor cells in inflammatory diseases such as inflammatory bowel disease136. The signaling events associated with this IL-6/gp130 signaling include activation of signaling proteins STAT3 and ribosomal protein S6
(RPS6)136. As we had recently reported RPS6 activation in models of endocrinopathic laminitis, investigating a similar signaling mechanism in sepsis was of great interest37.
Therefore, we hypothesized that lamellar failure in sepsis-related laminitis was related to IL-6/GP130-related signaling resulting in activation of STAT3 and RPS6. We therefore investigated this signaling in the lamellae in the same study in which CDH was instituted 12 h after OF administration; we were interested in determining if lamellar RPS6 and STAT3 activation was induced in sepsis-related laminitis, and if so, whether the activation was affected by CDH.
59 4.2 Materials and Methods
Animal Protocol
All horses in the OF group developed pyrexia (rectal temperature>38.5°C) within 18h of the oligofructose bolus, with the exception of one study subject who was administered 2.0g phenylbutazone IV at 10h due to lameness. All horses in the OF group developed tachycardia (>45 beats/min) within 16h of the oligofructose bolus.
Pedometer data in OF study subjects revealed a significant increase in limb movement of the ambient limbs (P<0.05) when compared to limbs subjected to controlled digital hypothermia.
Protein Extraction
Protein was extracted from the snap-frozen lamellar tissue of the four groups of study subjects (AMB CON, AMB OF, ICE CON, ICE OF). All samples were pulverized and homogenized in M-PER lysis buffer, protease and phosphatase inhibitor cocktails, and
PMSF. Samples were incubated on ice for 20 minutes, after which they were centrifuged and the supernatant collected and the protein concentration of the supernatant was quantified by the Bradford method. Samples were aliquotted and then stored at -80° C until use.
Western Blot Hybridization
Lamellar protein from AMB CON, AMB OF, and ICE OF study subjects was assessed via Western blot analysis for concentration of phosphorylated RPS6 (240/244), RPS6
(235/236), Akt, ERK, STAT1 (Ser727), STAT1 (Tyr701), STAT3 (Ser727), STAT3
(Tyr705), p38 MAPK, p70S6K, SAPK, and JAK2. Western blots were limited to analysis of only three study groups (AMB CON, AMB OF, and ICE OF) due to the
60 size of the gel apparatus. Antibodies recognizing the protein of interest irrespective of its phosphorylation state were also used to assess total protein concentrations.
Concentrations of β-actin were assessed in all samples to allow for normalization.
Each protein sample (20µg) was denatured, separated on a 4-15% gradient SDS-PAGE
GEL, and transferred to a PVDF membrane. 5% BSA in TBST was used to block the membranes for 1 hour at room temperature, and the blots were then probed with primary antibody against the protein of interest and incubated at 4°C overnight. After washing, the membranes were then incubated with goat anti-rabbit IgG secondary antibody coupled to horseradish peroxidase for 1 hour at room temperature. The membrane was then washed and the chemiluminescent signal was developed using west femto substrate. Band intensity was measured using image J software, and signal strength was normalized using β-actin band intensity.
Immunofluorescence
Lamellar sections frozen in OCT from AMB OF and ICE OF horses were used to immunolocalize the presence of both phosphorylated and total RPS6 and p70S6K. Ten-
µm cryosections were prepared from frozen lamellae and fixed for 15 minutes in 4% formaldehyde, then blocked in PBS containing 5% normal goat serum and Triton X-
100 for 1h at room temperature, then incubated at 4°C overnight with primary antibody. After 3 washes, slides were incubated with Alexafluor flourochrome conjugated secondary antibody for 1.5 hours. After this incubation, the slides were washed and cured overnight with a mounting media containing DAPI, a nuclear counterstain. The slides were then imaged on a Leica DM IRE laser assisted confocal microscope equipped with digital imaging software.
61 4.3: Results
Induction of sepsis in study subjects
All horses in the OF group developed pyrexia (rectal temperature>38.5°C) within 18h of the oligofructose bolus, with the exception of one study subject who was administered 2.0g phenylbutazone IV at 10h due to lameness. All horses in the OF group developed tachycardia (>45 beats/min) within 16h of the oligofructose bolus.
Pedometer data in OF study subjects revealed a significant increase in limb movement of the ambient limbs (P<0.05) when compared to limbs subjected to controlled digital hypothermia.
Effect of induction of sepsis related laminitis and digital hypothermia on growth factor signaling proteins, evaluated by western blot hybridization
AMB CON vs AMB OF
Analysis of the ratios of phospho-protein/β-actin concentrations of individual samples revealed marked increases in the phosphorylation states of AKT, ERK, p70S6K, RPS6
(240/244), RPS6 (235/236), STAT3 (Ser727), STAT3 (Tyr705), STAT1 (Ser727), and
STAT1 (Tyr701) in lamellar tissue from AMB OF limbs when compared to the lamellar tissues from AMB CON limbs. Analysis of the ratios of total protein/β-actin concentrations of the individual samples revealed a decrease in the total AKT in the lamellar tissues from AMB OF limbs (Figures 7,8,9).
AMB CON vs ICE OF
Analysis of the ratios of phospho-protein/β-actin concentrations of individual samples revealed no increases in the phosphorylation states of p70S6K and RPS6 in lamellar tissue from ICE OF limbs when compared to the lamellar tissues from AMB CON
62 limbs. Analysis of the ratios of total protein/β-actin concentrations of individual samples revealed a decrease in total RPS6 in lamellar tissues from ICE OF limbs
(Figures 7,8,9).
ICE OF vs AMB OF
Analysis of the ratios of phospho-protein/β-actin concentrations of individual samples revealed decreases in the phosphorylation states of RPS6(235/236), RPS6(240/244) P- p70S6K(T421/S424), and STAT3(s727) in the lamellar tissue from ICE OF limbs when compared to the lamellar tissues from AMB OF limbs. Analysis of the ratios of total protein/β-actin concentrations of individual samples did not reveal differences in any of the assessed mediators (Figures 7,8,9).
63
Figure 7: Western blots exhibiting phosphorylation of STAT3 (Y705) and STAT3 (S727), as well as quantitative data of the relative intensity of phosphorylation.
64
Figure 8: Western blots exhibiting phosphorylation of P-RPS6 (325/236), as well as quantitative data of the relative intensity of phosphorylation.
Figure 9: Western blots exhibiting phosphorylation of p70S6K, as well as quantitative data of the relative intensity of phosphorylation.
65
Figure 10 Immunohistochemistry comparing phosphorylation of RPS6 of AMB OF vs ICE OF limbs. Note the localization of the phosphorylated RPS6 to the edges of the secondary epidermal lamellae, indicating activity in the lamellar basal epithelial cell.
Figure 11 Immunohistochemistry comparing phosphorylation of STAT3 of AMB OF vs ICE OF limbs. Note the localization of the phosphorylated STAT3 to the edges of the secondary epidermal lamellae, indicating activity in the lamellar basal epithelial cell.
66
Localization of growth factor signaling proteins using immunofluorescence
Immunofluorescence comparing RPS6/p-RPS6 and STAT3/p-STAT3 revealed presence of RPS6 and STAT3 in the dermis and epidermis of the lamellae, while their phosphorylated counterparts (p-RPS6 and p-STAT3) were localized to the lamellar epithelium, indicating their presence in the lamellar basal epithelial cells (Figure 10,
11).
4.4 Discussion
These data reveal not only activation of growth factor signaling pathways in sepsis related laminitis, but perhaps more importantly a profound inhibition of these pathways through the use of controlled digital hypothermia.
Similar to the results of a recent study investigating growth factor signaling pathways in EMSAL using the EHC model, activation of growth factor signaling pathways in the lamellar tissues in which sepsis related laminitis was induced was clearly demonstrated by the increases in phosphorylated (activated) AKT, ERK, p70S6K, RPS6, and STAT3 in lamellar tissue from AMB OF limbs when compared to the lamellar tissues from AMB CON limbs37. Investigations of growth factor signaling in EMSAL have focused on signaling pathways downstream of insulin-like growth factor-1 receptor (IGF-1Rc), which is not only reported to be expressed in lamellar epithelial cells, but also has been widely studied in investigations into human tumor biology36,137. Signaling pathways downstream of IGR-1Rc, which are reported to be
67 well conserved amongst mammalian species, include the activation of two main signaling pathways: Ras/Raf/ERK 1/2 and P13K/AKT 138. Recent work in our laboratory demonstrated the activation of both of these main signaling pathways in the
EHC model of EMSAL, and activation of the ERK1/2 pathway in the CHO model of
SRL37,139. Interestingly, treatment with CDH in the CHO model did not result in a decrease in activation of the ERK1/2 pathway, indicating that the lamellar protection conferred by hypothermia in these study subjects was not through inhibition of the
ERK1/2 arm of growth factor signaling139. Similar results were found in the current study in which, although there was an increase in phosphorylation of ERK 1/2 in the lamellar tissue from study subjects administered oligofructose (AMB OF), there was no decrease in phosphorylation in the ICE lamellar tissue. The pattern of phosphorylation within the P13K/AKT pathway (represented by p70S6K and AKT) in the OF lamellar tissue in the current study indicates that, similar to study subjects in which laminitis is induced using the EHC model, there is activation of the P13K/AKT growth factor signaling pathway in sepsis related laminitis37. Perhaps of more clinical relevance is the fact that not only was the P13K/AKT pathway activated in this OF model of SRL (similar to the EHC model of EMSAL), CDH resulted in profound inhibition of activation of this pathway, as indicated by the marked decrease in phosphorylated p70S6K and RPS6 in ICE OF versus AMB OF lamellar tissue.
Interestingly, similar activation of growth factor signaling (specifically RPS6) has been implicated in the early stages of human neoplasia24,25,140,141. The epithelial to mesenchymal transition of a neoplastic cell bears marked similarities to the ultrastructural changes observed in the laminitic lamellar basal epithelial cell including
68 cytoskeletal dysregulation and dysadherence from the basement membrane14,22,38-40. In the case of neoplasia, adherence of epithelial cells to the basement membrane by hemidesmosomes and anchoring filaments is critical to maintenance of the epithelial phenotype; in equine laminitis, this adherence is the lynchpin of the suspensory apparatus of the distal phalanx142.
The similarities between epithelial to mesenchymal transition of a neoplastic epithelial cell and the morphologic changes in the lamellar basal epithelial cell are interesting not only from a pathophysiologic standpoint, they also allow for the consideration of new treatment modalities. Although it is the only therapy scientifically proven to prevent the development and progression of sepsis related laminitis, digital hypothermia is time and labor intensive, and if done continuously, it usually requires hospitalization88. The mammalian target of rapamycin complex 1 (mTORC1), represents a therapeutic target which is downstream of P13K/AKT and upstream of
P70S6K and RPS6 (both of which experienced a decrease in activation in the current study), implying that it could confer the same protective effects as digital hypothermia. mTORC1 is commonly blocked by rapamycin (a macrolide antibiotic) to prevent progression of malignancy in human neoplasia by inducing tumor cell apoptosis and suppressing angiogenesis143-145. Rapamycin’s widespread use in human oncology has a well-described side effect profile, but its use has never been described in the equine species146. Additionally, the high levels of complexity of the growth-factor signaling pathways, coupled with the dynamic nature of cellular signaling demonstrated in septic equine lamellar tissue, indicate that a thorough investigation of the suppression of
69 mTOR in the equine patient be conducted before rapamycin is widely accepted as a broadly applicable therapy for equine laminitis.
70
Chapter 5: Discussion
5.1 Inflammation and Sepsis-Related Laminitis
This series of investigations began with the presumption that, because there was a marked and repeatable increase in gene expression of a broad range of inflammatory mediators in models of SRL, the protection conferred by hypothermia would be due to a decrease in gene expression of these mediators. This was not a complete leap: a recent manuscript from our laboratory demonstrated that in the OF model of SRL with application of immediate hypothermia (digital hypothermia initiated at the same time as OF administration), the lamellar tissue subject to digital hypothermia exhibited a profound decrease in gene expression of a broad range of pro-inflammatory mediators
(CXCL-1, CXCL-8, IL-1β, COX-2, ICAM-1, E-selectin, and IL-6)89. This idea of an anti-inflammatory mechanism for cryoprotection has also been supported in other tissues such as skeletal muscle, lung, and neuronal tissue in a variety of species, all of which exhibited a decrease in inflammation with the institution of therapeutic hypothermia123,147-150.
In direct contrast to this previous work, however, the first study (Chapter 2) investigating the effect of delayed hypothermia (hypothermia not initiated until 12 hours post OF administration) on gene expression of inflammatory mediators not only found no inhibitory effect on the inflammatory profile of the lamellar tissue, but also detected an increased expression of leukocyte adhesion molecules in the ICE tissue.
71 This manuscript essentially uncoupled the ideas of inflammation and cryoprotection, but the late harvest point of the tissue (likely OG4 with severely damaged lamellar tissue) called into question the relevance of the results to the clinical patient.
In an effort to confirm or refute the findings of the delayed hypothermia model, an additional experiment was conducted in which the hypothermia was still instituted at a timepoint approximating the treatment of a clinical patient (12 hours post-OF bolus), but the tissue was harvested at an earlier timepoint (24 hours after the institution of digital hypothermia) to avoid evaluation at a timepoint with severe lamellar structural damage. The importance of evaluating the inflammatory profile of the lamellar tissue in SRL has been highlighted by investigations in the BWE, starch overload, and OF bolus models of SRL. The BWE model of SRL demonstrates a robust early inflammatory response, whereas the CHO overload models of SRL have been shown to have a modest early inflammatory response escalating to a marked pro- inflammatory state by the onset of lameness (OG1 laminitis)72,151. By OG3 laminitis in the CHO overload model, however, this inflammatory response has waned151.
Therefore, this temporally variable nature of lamellar inflammation in SRL called into question the validity of the findings in the study with an OG4 harvest timepoint, leading to the development of the second experiment which still utilized the clinically applicable delayed hypothermia timepoint for institution of therapy, but harvested the lamellar tissue after only 24 hours of hypothermia (36 hours after administration of
OF).
Results from this second experiment closely paralleled the results from the delayed hypothermia/late harvest experiment: the only 2 mediators which exhibited a
72 decrease in expression with the institution of digital hypothermia were IL-6 and COX-
2. These findings served to effectively redirect investigative efforts: since the cryoprotection conferred by hypothermia was clearly and repeatedly not through broad inhibition of inflammatory mediators, a more targeted investigation was initiated into signaling pathways which were decreased in hypothermic tissue (IL-6 cytokine family), as well as signaling mechanisms which have been demonstrated to be phosphorylated in other types of laminitis, namely growth factor signaling.
5.2 Growth Factor Signaling and Laminitis
The application of EMSAL research findings to SRL models is legitimized by the marked similarities seen in the ultrastructural changes to the secondary epidermal lamellae and lamellar basal epithelial cell in both types of laminitis. Namely, both the
EHC and OF models of laminitis have induced elongation and stretching of secondary epidermal lamellae, decrease in or complete dissolution of hemidesmosomes, and dysadherence of the lamellar basal epithelial cell from the basement membrane in study subjects7,11,14,27.
Recent work in our laboratory demonstrated an increased phosphorylation within IGF-1R pathways (both ERK1/2 and P13K arms) in the EHC model of EMSAL, and phosphorylation of the ERK1/2 pathway in the CHO model of SRL37,139.
Interestingly, the institution of digital hypothermia did not result in a decrease in phosphorylation of the ERK1/2 pathway in the CHO model of SRL, indicating that the
P13K signaling pathway is more likely to play a role in the cryoprotective effects of digital hypothermia in SRL37,139. The administration of OF in the current study induced phosphorylation of both the ERK1/2 and P13K arms of the IGR-1R pathway,
73 with an increase in most downstream effectors (AKT, ERK, p70S6K, RPS6 (240/244),
RPS6 (235/236)), as well as STAT1 and STAT3 (Figure 12). Importantly, the results from Chapter 5 demonstrated that similar signaling occurs in two distinct types/models of laminitis: the OF bolus model of SRL and the EHC model of EMSAL. Most notable, however, was the profound decrease in phosphorylation of p70S6K, RPS6, and AKT in the lamellar tissue subject to CDH, indicating that the cryoprotection conferred by hypothermia in SRL may be due to inhibition of the PI3K/mTOR arm of the IGF-1R signaling pathway (Figure 13).
Figure 12: Representation of the phosphorylation (activation) of downstream effectors of mTORC1 in SRL.
Figure 13: Representation of the lack of phosphorylation (activation) of the downstream effectors of mTORC1 in the limbs subject to digital hypothermia
74
Interestingly, IL-6, one of the only mediators to be consistently increased in both EHC and CHO overload laminitis models (and to be suppressed by digital hypothermia in said models) is also implicated in neoplastic and inflammatory disease through its activation of MAPK, PI-3K, and STAT3 pathways136. IL-6, a potent pro- inflammatory cytokine which mediates a wide variety of physiological functions, has provided a link between chronic inflammation and neoplasia since the 1960s when it was observed that the administration of inflammatory substances to wild-type mice resulted in murine plasma cell tumors, whereas IL-6 knockout mice were resistant to inflammation induced PCT formation152,153. Activated downstream of IL6 via gp130 and JAK2, STAT3 serves to activate an array of pro-apoptotic and proliferative pathways and is the most commonly observed member of the STAT family to be present in a constitutively active state in many types of neoplasia including lung, breast, renal, prostate, ovarian, and cervical cancer153. A recent study investigating the role of both STAT3 (a downstream effector of IL-6) and mTORC1 in mice expressing excessive GP130/STAT3 activation (a gastric tumorigenesis model) found that activation of the gp130 receptor by IL-6 simultaneously activates the STAT3 and
PI3K/mTORC1 pathways within neoplastic cells136. Activation of these pathways, in turn, promoted the development of gastric tumors in the above-described murine model136.
Two downstream effectors of mTORC1, specifically p70S6K and RPS6, have been implicated in cytoskeletal reorganization similar to that seen in the intermediate filament reorganization of the laminitic LBEC. Ovarian cancer cells in which p70S6K
75 was constitutively expressed and activated in vitro developed actin-rich projections including lamellipodia and filopodia, while no such cytoskeletal changes were noted in the untransfected controlled cells154. Similarly, activation of RPS6 (downstream effector of both mTORC1 and P70S6K) was recently shown to occur during breakdown of the basal ectoplasmic specialization, a tightly packed actin filament bundle which is vital to the integrity of the blood testis barrier155. Further strengthening the link between mTORC1 signaling and cytoskeleton re-organization, rapamycin (a potent mTOR inhibitor) was found to prevent IGF-1 stimulated F-actin reorganization in human rhabdomyosarcoma, Ewing sarcoma, glioblastoma, and prostate carcinoma cells in vitro156. Further investigation into the underlying mechanism of the inhibitory effects of rapamycin on cytoskeletal reorganization and cell motility demonstrated down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 pathways157. Although the exact mechanism of the changes in cytoskeletal structure seen in the lamellar basal epithelial cells has yet to be elucidated, the strong association between mTORC1 pathways and actin filament rearrangements in human neoplasia, when considered alongside the inhibition of downstream effectors of mTORC1 (RPS6 and P70S6K) in the digital hypothermia models of laminitis, offer a clear argument in favor of an IL6/gp130/mTORC1/RPS6 pathway for the damage to lamellar basal epithelial cells.
Growth factor signaling pathways have been implicated in epithelial to mesenchymal transition in human epithelial cancers and inflammatory diseases, in which cytoskeletal dysregulation bears a striking resemblance to the loss of hemidesmosomal integrity and cytoskeletal changes observed in both EMSAL and
76 SRL study subjects. Specifically, both laminitis study subjects and neoplastic epithelial cells undergoing epithelial to mesenchymal transition undergo loss of: 1) cell-cell adhesion complexes (HDs), 2) attachment of cells to the basement membrane, and 3) in the case of neoplastic epithelial cells, loss of attachment of the intermediate filaments to the inner plaque of the hemidesmosome7,9. In particular, the loss of the anchoring effect of the intermediate filaments on the cell nucleus results in a more basal location of the LBEC nucleus (in the case of laminitis patients), and rounding of the nucleus itself18. In the case of epithelial to mesenchymal transition (EMT), the hemidesmosomal protein integrin α6 has been localized in the lamellipodia and filopodia of migrating cpithelial carcinoma cells, demonstrating its pivotal role in the proliferation and metastasis of neoplastic cells158. Although the lamellar basal epithelial cells obviously do not progress to proliferation and metastasis, the striking similarities between cytoskeletal changes occurring in early EMT and laminitis allow for ready comparison of both pathophysiology and potential treatment for the two diseases. Importantly, the discovery that the cryoprotection conferred by digital hypothermia could be through inhibition of downstream effectors of mTOR opens up new targeted therapeutic options for prevention of laminitis such as rapamycin, an mTOR inhibitor which has been used to mitigate growth and metastasis of human neoplasia25,143,159,160.
The body of work outlined above, along with recent work in our laboratory, has established, for the first time, convergence of the same signaling in models of all three types of laminitis: SRL, EMSAL, and SLL. Previous work in EMSAL demonstrated
RPS6 activation in the EHC model37; the data in Chapter 5 demonstrates
77 phosphorylation of STAT3 and RPS6 in the OF model of SRL; and recent work in our laboratory discovered similar activation of both STAT3 and RPS6 in the unilateral weight-bearing model of SLL (Belknap lab, unpublished data). The importance of this common signaling pathway for three disparate clinical presentations cannot be overstated, especially since therapeutic hypothermia was demonstrated to decrease phosphorylation of the downstream effectors of these pathways in the SRL model.
Future investigations will include assessment of the efficacy of digital hypothermia in both the EHC model of EMSAL and the unilateral weight-bearing model of SLL.
Similar parallel activation of RPS6 and STAT3 has been observed in human gastrointestinal cancers, and this coactivation of mTORC1 and STAT3 in tumor cells was linked to gp130 ligation by the IL-6 family cytokines in a murine inflammation- associated gastrointestinal tumor model136. Interestingly, inhibition of phosphorylation of mTORC1 through the use of RAD0001 (everolimus) suppressed tumorogenesis in murine models of both colitis associated cancer and gastric carcinoma136. Furthermore, following everolimus therapy, the phosphorylation of the tyrosine moiety of STAT3 remained unaffected in tumor cells, while the phosphorylation of mTORC1 target
RPS6 was markedly decreased during the course of therapy and returned after the cessation of everolimus administration. It is possible that, had the investigators examined the effect of therapy on the serine moiety of STAT3, they would have noticed a decrease in phosphorylation, since this was observed in our investigations.
Although STAT3 has long been implicated in inflammation associated neoplasias
161,162, this study indicated that gp130-dependent activation of PI3K/mTORC1 pathway is required for inflammation-associated gastrointestinal tumorigenesis, opening the
78 door for the use of PI3K/mTORC1 inhibitors in the treatment of these neoplasias136.
Since our lamellar basal epithelial cells displayed not only a similar phosphorylation signature (simultaneous activation of RPS6 and STAT3), but also the same set of early cytoskeletal abnormalities seen in epithelial cancers, it follows that gp130 dependent activation of the PI3K/mTORC1 pathway may be a key component of the pathophysiology of all 3 types of laminitis. Furthermore, treatments which have been effective in halting tumor progression in human epithelial neoplasia, namely mTORC1 inhibitors, may have potential to mitigate the debilitating lamellar damage seen in sepsis related, endocrinopathic, and supporting limb laminitis.
79
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