Effect of digital hypothermia on lamellar inflammatory signaling in the euglycemic hyperinsulinemic clamp laminitis model

Abstract Background Continuous digital hypothermia (CDH) prevents lamellar failure in the euglycemic hyperinsulinemic clamp (EHC) model of laminitis, but the protective mechanisms are unclear. Hypothesis/Objectives To determine if CDH inhibits lamellar inflammatory signaling in the EHC model of laminitis. Animals Eight Standardbred horses. Methods Prospective experimental study. Horses underwent an EHC, with 1 forelimb treated with CDH and the other kept at ambient temperature (AMB). Horses were euthanized 48 hours after initiation of the EHC and lamellar tissue was analyzed via polymerase chain reaction (pro‐inflammatory cytokine and chemokine genes—CXCL1, CXCL6, CXCL8, IL‐6, MCP‐1, MCP‐2, IL‐1β, IL‐11, cyclooxygenase 1 and 2, tumour necrosis factor‐alpha [TNF‐α], E‐selectin, and intercellular adhesion molecule‐1 [ICAM‐1]) and immunoblotting (phosphorylated and total signal transducer and activator of transcription 1 [STAT1] and STAT3). Results Compared to AMB, lamellar messenger ribonucleic acid (mRNA) concentrations of CXCL6 (P =.02), CXCL8 (P = .008), IL‐6 (P = .008), IL‐1β (P = .008), IL‐11 (P = .008), and cyclooxygenase‐2 (P = .008) were decreased in CDH. Cyclooxygenase‐1 (P = .008) was increased in CDH, while CXCL1 (P = .15), MCP‐1 (P = .05), MCP‐2 (P = .46), TNF‐α (P = .05), E‐selectin (P = .15), and ICAM‐1 (P = .15) mRNA were not significantly different. Compared to AMB, lamellar concentration of total STAT3 protein was decreased in CDH (P < .001), but there was no change in phosphorylated STAT3 (P‐STAT3 [S727] P = .19; P‐STAT3 [Y705] P = .05). There was no change in lamellar concentrations of total STAT1 (P = .75) or phosphorylated STAT1 (P‐STAT1 [S727], P = .25; P‐STAT1 [Y701], P = .64). Conclusions and Clinical Importance These data add further support for the use of CDH as a first aid treatment for severe acute laminitis associated with hyperinsulinemia in horses.

Conclusions and Clinical Importance: These data add further support for the use of CDH as a first aid treatment for severe acute laminitis associated with hyperinsulinemia in horses. Recent research on the pathogenesis of endocrinopathic laminitis has focused on lamellar vascular derangements, 5,6 disturbances of glucose metabolism, 7,8 and lamellar events related to insulin-like growth factor-1 receptor (IGF-1R) activation including cellular proliferation and loss of cell adhesion. 9,10 A role for IGF-1R signaling has been supported by the presence of IGF-1R on lamellar epithelial cells, 10,11 downregulation of lamellar IGF-1R expression in horses undergoing an EHC, 12 and also evidence of activated signaling pathways related to IGF-1R (ie, the mammalian target of rapamycin complex-1 [mTORC1]/ribosomal protein S6 [RPS6] signaling pathway). 9 A recent study showed that insulin does not readily bind to lamellar IGF-1R at relevant concentrations, 13 questioning whether direct activation of IGF-1R by insulin is responsible for activating lamellar signaling pathways related to IGF-1R. 9 Lamellar leukocyte infiltration 3 and increased lamellar gene expression of pro-inflammatory cytokines 14 has been observed in the EHC model, suggesting a role for lamellar inflammation in the pathogenesis of insulin-associated laminitis.
Inflammation also provides an alternative, indirect explanation for activation of signaling pathways related to IGF-1R in the EHC model, for example, via inflammatory mediators including interleukin-6 (IL-6) through the gp130 receptor. 15 It is well established that CDH reduces lamellar injury in sepsisrelated laminitis and this protection has been partly attributed to an anti-inflammatory effect. 16,17 In support of this, CDH has been shown to attenuate lamellar inflammation in sepsis-associated laminitis models by reducing inflammatory cell infiltration 18 and the expression of pro-inflammatory cytokines, chemokines, cyclooxygenase-2, and endothelial adhesion molecules. 16 In addition, the effects of CDH on lamellar inflammatory signaling pathways has helped identify signaling pathways that could be exploited therapeutically. 15,17 It is currently unclear if CDH affects lamellar inflammation in the EHC model of endocrinopathic laminitis. We hypothesized that CDH would have a similar inhibitory effect on lamellar inflammation to that observed in sepsis-associated laminitis models. 16,17 To investigate this, the messenger ribonucleic acid (mRNA) concentration of inflammatory mediators (pro-inflammatory cytokines, chemokines, endothelial adhesion molecules, and cyclooxygenase enzymes) and protein concentration of inflammation-associated proteins (STAT proteins) were quantified in the lamellar tissue of CDH-treated and untreated feet from horses undergoing the EHC model of endocrinopathic laminitis.

This project was approved by the University of Queensland Animal
Ethics Committee 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.

| Animal protocol
Eight healthy Standardbred geldings (mean age: 6.3 ± 1.7 years; mean bodyweight: 447.8 ± 36.9 kg) recently retired (<4 weeks) from racing underwent laminitis induction using the EHC model. All horses were sound at the walk and had no gross or radiographic abnormalities of the feet. For the duration of the experiment, all horses were confined to stocks and received ad libitum access to alfalfa hay and water. An EHC was performed for 48 hours as previously described. 3 Briefly, an intravenous bolus (45 mIU/kg bwt) of recombinant human insulin (Humulin-R, Eli-lily, West Ryde, Australia) diluted in 50 mL of 0.9% sodium chloride (Baxter, Old Toongabbie, Australia) was administered via a 14-gauge jugular catheter and was immediately followed by a continuous intravenous infusion of insulin in 0.9% sodium chloride (Baxter) at a rate of 6 mIU/(kg bwt min). A continuous intravenous infusion of 50% glucose (Baxter) was administered concurrently, with the administration rate adjusted to maintain euglycemia (4.0 ± 1.0 mM/L). Blood glucose was measured using a portable glucometer (Roche Diagnostics, Indianapolis, Indiana) every 5 minutes until euglycemia was achieved for 30 minutes without the need to change the glucose infusion rate. Blood glucose was then measured every 30 minutes for the remainder of the experiment. For the duration of the experiment, horses were constantly monitored and vital signs were measured every 2 hours.
Thirty minutes before starting the EHC, 1 forelimb was randomly selected by coin toss to be cooled for the duration of the 48 hour EHC using a rubber boot (Bigfoot Ice boots, Esk, Australia) containing ice cubes and water. Ice was replenished as required to maintain a 50% ice, 50% water mixture to a level just distal to the carpus. The contralateral limb was kept at ambient temperature (AMB). Hoof wall surface temperature in CDH and AMB limbs was monitored using hoof wall thermistors attached to data-logging devices as previously described. 16 After 48 hours of the EHC, the horses were euthanized with pentobarbital sodium (20 mg/kg bwt IV). Dorsal lamellar tissue samples were rapidly dissected from the hoof and distal phalanx in each forelimb (AMB and CDH) from each study subject and were snap frozen in liquid nitrogen .

| RNA isolation and complementary DNA synthesis
Three separate sections of lamellar tissue from each forelimb of each horse were pulverized and total RNA was isolated using a guanidine thiocyanate spin column method (Agilent Technologies, West Cedar, Texas) with a DNase treatment to remove genomic DNA contamination. Messenger RNA was then isolated with a poly-A tail streptavidin magnetic bead kit (Roche Diagnostics) and used to make complementary DNA (cDNA) using a total of 400 ng of mRNA. The cDNA was diluted at 1 : 5, 1 : 50, and 1 : 500 and frozen at −20 C until used for real-time quantitative polymerase chain reaction (RT-qPCR) analysis.

| Real-time polymerase chain reaction
Real-time quantitative polymerase chain reaction was performed using a thermocycler (Roche LC 2.0; Roche Life Science, Indianapolis, Indiana) as previously described. 14 Each set of reactions containing AMB and CDH lamellar tissue was run in duplicate and included double distilled water as a negative control, controls without added reverse transcriptase, and a standard curve made from serial dilutions were entered into a computer program (geNorm) to test the suitability of each gene as a housekeeping gene for the lamellar tissue samples.
Both genes were determined to be satisfactory by the program and a geometric mean was obtained from the 2 genes' data in order to generate a normalization factor for each lamellar tissue sample. The amplification data for the genes of interest were then divided by the unique normalization factor of the selected housekeeping genes in each lamellar tissue sample, creating a normalized copy number value.
The data are reported as either absolute copy number or fold-change from AMB values (calculated by dividing the normalized CDH expression values by the normalized AMB values for each gene).

| Protein extraction
Protein was extracted from snap-frozen lamellar samples as previously described. 9,14 Briefly, all samples underwent an initial pulverization step before homogenization on ice in a commercially available lysis buffer (Thermo Scientific, Waltham, Massachusetts) with the addition of protease and phosphatase inhibitors and phenylmethylsulfonyl fluoride. Samples were then incubated on ice for 30 minutes. Supernatant was separated by centrifugation, and protein concentration was quantified using the Bradford method. Protein samples were stored at −80 C until analysis.

| Immunoblotting
The concentration of phosphoprotein was assessed as previously described 14 via Western immunoblot analysis of lamellar samples using commercially available antibodies raised against STAT1 P-(S727), STAT1 P-(Y701), STAT3 P-(S727), and STAT3 P-(Y705). Total STAT1 and STAT3 protein concentrations were assessed using antibodies recognizing the protein of interest regardless of its phosphorylation state as previously described. 14

| Western immunoblotting
Lamellar concentration of total STAT3 protein was decreased in CDH limbs compared to AMB limbs (P < .001; Table 2

| DISCUSSION
In a recent study in Standardbred horses that underwent the EHC model of laminitis, lamellar mRNA concentrations of inflammatory  16 In the OF model, CDH decreased lamellar mRNA concentrations of the same inflammatory mediators observed to be decreased in our study. 16 Importantly, it is thought that the anti-inflammatory effects of CDH help to reduce the severity of acute lamellar injury in sepsis. 16 Furthermore, it has been suggested that cell signaling mechanisms upstream of lamellar inflammatory mediators could represent potential pharmacological targets 15,16 particularly IL-6/gp130 signaling, which may be activated in the EHC and OF laminitis models. 15 Indeed, the decreased lamellar mRNA concentration of IL-6 observed in CDH limbs in our study supports that inhibition of IL-6/gp130 signaling could be associated with reducing the severity of lamellar injury through inhibition of mTORC1 and STAT3 activation. 9,15,17 STAT1 and STAT3 play an important role in the development of inflammatory disease by serving as transcription factors, which result in the production of various inflammatory mediators. 19,20 Evidence of STAT3 activation has been observed in the EHC 14 and OF laminitis models 15 and it has been hypothesized that STAT3 is activated via IL-6/gp130 signaling. 15 Furthermore, it has been suggested that STAT3 activation is associated with loss of lamellar epithelial cell adhesion molecules (ie, desmosomes) in the EHC model and that inhibition of STAT3 activation via mTORC1 inhibition could protect the epidermal lamellae from structural failure. 15 Our study found that lamellar concentrations of activated STAT1 and STAT3 were not significantly different between CDH and AMB limbs, suggesting that this might not be a major therapeutic mechanism of CDH in the EHC model. However, total STAT3 concentrations were significantly lower in CDH limbs compared to AMB limbs, which is consistent with downregulation of STAT3 expression in CDH limbs. This finding could be associated with the decrease in IL-6 lamellar mRNA concentrations observed in our study, as IL-6 induces STAT3 gene expression. 21,22 COX-2 is induced by pro-inflammatory cytokines, growth factors, and oncogenes, and its overexpression is associated with the inflammatory response and carcinogenesis. [23][24][25][26][27] Lamellar mRNA concentration of COX-2 is markedly increased in the EHC and OF laminitis models consistent with COX-2 overexpression because of local inflammatory response and/or growth factor signaling. 14,16,28 Furthermore, it has been hypothesized that overexpression of COX-2 could play an important role in the pathogenesis of laminitis by potentiating epithelial-to-mesenchymal transition, 28 an event that has been linked to the pathogenesis of laminitis in association with activation of both p70S6 kinase and RPS6 downstream of the mTORC1 signaling pathway. 9,15 While it has been suggested that COX inhibitors may represent a therapeutic target for the treatment and prevention of laminitis, 28,29 it is unknown whether the inhibition of lamellar COX-2 alone could prevent the development of insulin-induced laminitis. In our study, it was found that lamellar mRNA concentrations of COX-2 were lower in CDH limbs compared to AMB limbs, a finding also observed in the OF model. 17 IL-1β is a pro-inflammatory cytokine that is mainly produced by macrophages and fibroblasts (the predominant cell type in dermal lamellae) and increases the expression of other inflammatory mediators including COX-2, IL-6, and matrix metalloproteinases, which are associated with the degradation and remodeling of tissues. 30 Importantly, lamellar IL-1β expression has been shown to be increased in the EHC laminitis model. 14 33 Importantly, it has been noted that signaling pathways responsible for nutrient sensing and metabolism, including AMPK and mTOR, intersect with inflammatory pathways and therefore may influence each other in the EHC model. 14 Furthermore, inflammation has been shown to alter protein homeostasis and increase amino acid metabolic demands, 39,40 which could further contribute to the disruption of lamellar structural protein homeostasis (dysregulation of the synthesis and degradation of cell adhesion molecules, cytoskeletal proteins and extracellular matrix proteins) that causes stretch, loss of lamellar epithelial cell adhesion, and extracellular matrix degradation in laminitis. 3,35 Similarly, mechanical influences on tissue can stimulate the local expression of inflammatory mediators (particularly COX-2), as it has been shown in fibroblasts that cyclical stretch (and also the magnitude of stretching) increases COX-2 expression. 30,41 This suggests that unnatural mechanical forces acting on components of the lamellar tissue as a consequence of lamellar failure (stretch and separation) may also influence inflammatory gene expression.
A limitation of our study from a clinical perspective was that CDH was applied at the commencement of the EHC before the development of clinical signs of laminitis, and it remains unclear whether CDH applied during acute laminitis (initiated after the onset of lameness) would have a therapeutic effect. Interestingly, although such a protective effect was observed in the OF model when CDH was applied after lameness developed, 42 CDH did not inhibit increases in lamellar mRNA concentrations of inflammatory mediators, suggesting that this was not an important therapeutic mechanism during the acute phase of sepsis-related laminitis. 43 It is important to recognize, however, that the findings of that study as well as our study are limited by the fact that mRNA concentrations were investigated as opposed to quantification of peptides/proteins of interest in their biologically active forms. Therefore, the functional activity and role of inflammatory events could not be fully elucidated.
In conclusion, our study demonstrated that CDH inhibited lamellar inflammatory signaling in the EHC model of endocrinopathic laminitis. Further studies that investigate the cause, timeline, and role of lamellar inflammatory signaling in the pathogenesis of different forms of laminitis are warranted.

ACKNOWLEDGMENT
This study was supported by a Grayson-Jockey Club Research Foundation Grant.