Influence of digital hypothermia on lamellar events related to IL-6/gp130 signalling in equine sepsis-related laminitis.

BACKGROUND: Interleukin-6 (IL-6) is consistently increased in the digital lamellae in different studies of sepsis-related laminitis (SRL). IL-6 signalling through the gp130 receptor activates similar signalling (i.e. mTORC1-related signalling) previously reported to be activated in models of endocrinopathic laminitis. OBJECTIVES: To assess the activation state of signalling proteins downstream of IL-6/gp130 receptor complex activation in an experimental model of SRL. STUDY DESIGN: Randomised experimental study. METHODS: Lamellar phospho-(P) protein concentrations downstream of the IL-6/gp130 receptors were assessed in the oligofructose (OF) model of SRL. Fifteen Standardbred horses were administered water (CON, n = 8) or oligofructose (OF, n = 7) via a nasogastric tube. At 12 h post-OF/water administration, one randomly assigned forelimb was exposed to continuous digital hypothermia (CDH) by placement in ice water (ICE, maintained at <7°C); the other forelimb was maintained at ambient temperature (AMB). Lamellar tissue samples were collected after 24 h of CDH from both ICE and AMB forelimbs and immediately snap-frozen. Lamellar proteins of interest were assessed by immunoblotting and immunofluorescence. RESULTS: Immunoblotting revealed increase (P<0.05) in the phosphorylation states of Akt (Ser 473), RPS6 (Ser235/236), RPS6 (Ser240/244), STAT3 (Ser727) and STAT3 (Tyr705) in lamellar tissue from OF-treated animals (AMB OF vs. AMB CON limbs); CDH resulted in decreased (P<0.05) lamellar concentrations of phosphorylated Akt, p70S6K, RPS6 (235/236), RPS6 (240/244) and STAT3 (S727) in OF-treated animals (AMB OF vs. ICE OF). Immunofluorescence showed that activated/phosphorylated forms of RPS6 and STAT3 were primarily localised to lamellar epithelial cells. MAIN LIMITATIONS: The nature, sequence and timing of sub-cellular events in this experimental model may differ from those that accompany naturally occurring sepsis. CONCLUSIONS: There were increased lamellar concentrations of activated signalling proteins downstream of the IL-6/Gp130 receptor complex in OF-treated horses; CDH inhibited this activation for the majority of the proteins assessed. These results demonstrate similar lamellar signalling (e.g. mTORC1-related signalling) and, therefore, possible therapeutic targets occurring in sepsis-related laminitis as previously reported in models of endocrinopathic laminitis.

Continuous digital hypothermia prevents lamellar failure in the euglycaemic hyperinsulinaemic clamp model of equine laminitis.

BACKGROUND: Continuous digital hypothermia can prevent the development and progression of laminitis associated with sepsis but its effects on laminitis due to hyperinsulinaemia are unknown. OBJECTIVES: To determine the effects of continuous digital hypothermia on laminitis development in the euglycaemic hyperinsulinaemic clamp model. STUDY DESIGN: Randomised, controlled (within subject), blinded, experiment. METHODS: Eight clinically normal Standardbred horses underwent laminitis induction using the euglycaemic hyperinsulinaemic clamp model (EHC). At initiation of the EHC, one forelimb was continuously cooled (ICE), with the other maintained at ambient temperature (AMB). Dorsal lamellar sections (proximal, middle, distal) were harvested 48 h after initiation of the EHC and were analysed using histological scoring (0-3) and histomorphometry. Cellular proliferation was quantified by counting epidermal cell nuclei staining positive with an immunohistochemical proliferation marker (TPX2). RESULTS: Severe elongation and disruption of SEL with dermo-epidermal separation (score of 3) was observed in all AMB feet at one or more section locations, but was not observed in any ICE sections. Overall 92% of the AMB sections received the most severe histological score (grade 3) and 8% were grade 2, whereas ICE sections were classified as either grade 1 (50%) or grade 2 (50%). Relative to AMB feet, ICE sections were 98% less likely to exhibit grades 2 or 3 (OR: 0.02, 95% CI 0.001, 0.365; P<0.01). Histomorphometry measurements of total and nonkeratinised primary epidermal lamellar length were significantly increased (P<0.01) in AMB limbs compared with ICE. TPX2 positive cell counts were significantly increased (P<0.01) in AMB limbs compared with ICE. MAIN LIMITATIONS: Continuous digital hypothermia was initiated before recognition of laminitis and therefore the clinical applicability requires further investigation. CONCLUSIONS: Continuous digital hypothermia reduced the severity of laminitis in the EHC model and prevented histological lesions compatible with lamellar structural failure.

Ex vivo effects of insulin on the structural integrity of equine digital lamellae.

BACKGROUND: Laminitis has a considerable impact on the equine industry. Endocrinopathic laminitis is the most common form and affected horses often have hyperinsulinaemia due to an underlying metabolic disorder. OBJECTIVES: The aim of this study was to determine if insulin weakens the structural integrity of digital lamellae and to develop an ex vivo model for the study of hyperinsulinaemia-induced lamellar failure. STUDY DESIGN: Ex vivo experiment. METHODS: Biomechanical testing was used to assess the structural integrity of lamellar explants exposed to either medium alone (control) or medium supplemented with insulin. Lamellar explants comprised of hoof wall, lamellar tissue and distal phalanx were harvested from four adult horses with no evidence of inflammatory disease or pre-existing disease of the digit. Following an equilibration period, explants were incubated in medium or medium supplemented with insulin (2.5 µg/ml) for 8 h prior to biomechanical testing to obtain load (N), stress (MPa), elongation to failure (mm), and Young's modulus (MPa) for each explant. Significant differences were assessed using a mixed linear model with horses as a random factor and control or insulin-treated group as a fixed factor. RESULTS: Lamellar explants incubated in medium supplemented with insulin failed at significantly lower load (P = 0.0001) and lower stress (P = 0.001) and had greater elongation to failure (P = 0.02). MAIN LIMITATIONS: In addition to the ex vivo nature of the study, location-dependent variability in explant structural integrity and variable diffusion of nutrients due to explant size may have been limitations. However, the study design attempted to account for these limitations through random assignment of explants to treatment groups independent of location and by evaluating stress to failure. CONCLUSIONS: Insulin weakens the structural integrity of equine lamellar explants and an ex vivo model for evaluation of hyperinsulinaemia-induced lamellar failure was established. The summary is available in Spanish - see Supporting Information.

Effect of Continuous Digital Hypothermia on Lamellar Inflammatory Signaling When Applied at a Clinically-Relevant Timepoint in the Oligofructose Laminitis Model.

BACKGROUND: Although continuous digital hypothermia (CDH) protects lamellae from injury in the oligofructose (OF) model of sepsis-related laminitis (SRL), conflicting results exist from these studies regarding effects of CDH on lamellar inflammatory events. HYPOTHESIS/OBJECTIVES: To determine the effect of CDH on lamellar inflammatory events in normal and OF-treated horses when instituted at a clinically relevant time point (onset of clinical signs of sepsis in this model). ANIMALS: Standardbred geldings (n = 15) aged 3-11 years were used. METHODS: In a randomized, controlled discovery study, animals were administered either OF (OF group, n = 8) or water (CON group, n = 8) by nasogastric tube and CDH was initiated in one forelimb (ICE) 12 hours later. Lamellar tissue samples were collected 24 hours after initiation of CDH (ICE and ambient [AMB] forelimbs). Lamellar mRNA concentrations of inflammatory mediators and lamellar leukocyte numbers were assessed using qPCR and immunohistochemistry, respectively; values from four sample groups (CON AMB, OF AMB, CON ICE, and OF ICE) were analyzed using mixed model linear regression. RESULTS: Although lamellar mRNA concentrations of multiple inflammatory mediators (IL-1ß, IL-6, CXCL1, MCP2, COX-2) were increased after OF administration (OF AMB group versus CON AMB; P < 0.05), only 2 inflammatory mediators (IL-6 and COX-2) and lamellar leukocyte numbers were decreased with CDH (OF ICE versus OF AMB; P < 0.05). CONCLUSIONS AND CLINICAL IMPORTANCE: Continuous digital hypothermia initiated at a time point similar to that commonly used clinically (clinical onset of sepsis) resulted in a more focused inhibition of inflammatory signaling.

A novel model to assess lamellar signaling relevant to preferential weight bearing in the horse.

Supporting limb laminitis (SLL) is a devastating sequela to severe unilateral lameness in equine patients. The manifestation of SLL, which usually only affects one limb, is unpredictable and the etiology is unknown. A novel, non-painful preferential weight bearing model designed to mimic the effects of severe unilateral forelimb lameness was developed to assess lamellar signaling events in the supporting limb (SL). A custom v-shaped insert was attached to the shoe of one forelimb to prevent normal weight bearing and redistribute weight onto the SL. Testing of the insert using a custom scale platform built into the floor of stocks confirmed increased distribution of weight on the SL compared with the unloaded forelimb (UL) and the contralateral (CH) and ipsilateral (IH) hind limbs in six Standardbred horses. In a second part of the study, eight healthy Standardbred horses were fitted with the insert and tied with consistent monitoring and free access to hay and water for 48 h, after which the lamellae were harvested. Real-time qPCR was performed to assess lamellar mRNA concentrations of inflammatory genes and immunoblotting and immunofluorescence were performed to assess lamellar protein concentration and cellular localization of hypoxia-related proteins, respectively. Lamellar mRNA concentrations of inflammatory signaling proteins did not differ between SL and either CH or IH samples. HIF-1α concentrations were greater (P < 0.05) in the SL compared to the CH. This work establishes an experimental model to study preferential weight bearing and initial results suggest that lamellar hypoxia may occur in the SL.

Effect of Delayed Digital Hypothermia on Lamellar Inflammatory Signaling in the Oligofructose Laminitis Model.

BACKGROUND: In the oligofructose (OF) model of sepsis-related laminitis (SRL), digital hypothermia ("cryotherapy") initiated before the onset of clinical signs is reported not only to limit lamellar injury, but also to cause marked inhibition of lamellar inflammatory signaling. HYPOTHESIS/OBJECTIVES: Because hypothermia also has been reported to be protective when not initiated until the onset of lameness in the OF model of SRL, we hypothesized that the lamellar protection conferred by hypothermia is caused by local lamellar inhibition of inflammatory signaling as described when hypothermia was initiated earlier in the disease process. ANIMALS: Eight Standardbred geldings aged 3-11 years with no lameness and no abnormalities of the feet detectable by gross or radiographic examination. METHODS: Using the OF model of SRL, lamellar mRNA concentrations of proinflammatory cytokines, chemokines, and endothelial adhesion proteins were compared between samples from treated limbs (CRYO, submerged in ice water for 36 hour starting at the onset of lameness), untreated limbs (NON-CRYO, opposite limb from CRYO limbs maintained at ambient temperature), and untreated limbs from normal horses in which laminitis was not induced (CON). RESULTS: Although OF administration resulted in increases in lamellar mRNA concentrations of several inflammatory mediators in NON-CRYO limbs (vs CON), digital hypothermia had no significant effect on these increases. CONCLUSIONS AND CLINICAL IMPORTANCE: The lack of inflammatory inhibition in lamellar tissue samples in our study indicates that the protective effects of digital hypothermia instituted at the onset of clinical signs of laminitis do not arise from inhibition of inflammatory pathways.

Lamellar events related to insulin-like growth factor-1 receptor signalling in two models relevant to endocrinopathic laminitis.

BACKGROUND: Insulin dysregulation, obesity, and exposure to high-nonstructural carbohydrate (NSC) forage are risk factors for equine metabolic syndrome-associated laminitis (EMSAL); high systemic insulin concentrations in EMSAL are proposed to induce cellular dysregulation in the digital lamellae through activation of the insulin-like growth factor-1 receptor. OBJECTIVES: To use a dietary challenge model (DCM) and a euglycaemic-hyperinsulinaemic clamp (EHC) model to assess lamellar growth factor-related signalling. STUDY DESIGN: Lamellar phospho (P)-protein concentrations of signalling proteins important in growth factor-related signalling were assessed in 2 models: 1) lean and obese ponies on a low- or high-NSC diet; and 2) EHC model using Standardbred horses. METHODS: Ponies stratified for body condition (lean [LN, n = 11] and obese [OB, n = 11]) were exposed to a low-NSC diet (LO, n = 5 per group for LN LO and OB LO) or a high NSC diet (HI, n = 6 per group for LN HI and OB HI groups) for 7 days. For the EHC model, horses were administered insulin (constant rate infusion [6 mIU/kg bwt/min] combined with 50% dextrose, EHC group, n = 8)] or saline (0.57 mL/kg bwt/h, CON group, n = 8) for 48 h. Immunoblotting was employed to assess concentrations of activated/phosphorylated and total protein for members of the PI3K/Akt/mTORC1 and Ras/ERK pathways in lamellar samples from both models. RESULTS: In the DCM, lamellar P-(Ser 240/244) RPS6 was increased in OB HI ponies (vs. OB LO, P<0.05); positive correlations existed (P<0.05; r>0.5) between Day 7 basal serum insulin concentrations and lamellar concentrations of P-p70S6K and P-(Ser 240/244) RPS6. In the EHC model, lamellar concentrations of P-Akt, P-p70S6K, P-ERK 1/2, P-p90RSK, and both P-(Ser 235/236) and P-(Ser 240/244) RPS6 were increased in the EHC group (vs. CON, P<0.05). MAIN LIMITATIONS: The primary limitations of this study are the small number of animals per group in the DCM study, and the fact that many animals did not develop laminitis as that was not the endpoint of either study. CONCLUSIONS: These results support further investigation of mTORC1/RPS6 signalling as a potential therapeutic target(s) in EMSAL. The Summary is available in Chinese - see Supporting Information.

Mechanical nociceptive thresholds of dorsal laminae in horses after local anaesthesia of the palmar digital nerves or dorsal branches of the digital nerve.

We examined the hypothesis that the palmar digital nerves (PDNs), but not the dorsal branches (DBs) of the digital nerves, innervate the sensitive dorsal laminae of the equine foot by evaluating the effects of perineural anaesthesia of the PDNs and DBs separately on pain sensation evoked via mechanical stimulation of the dorsal laminae and other regions of the equine foot. Six clinically normal mares were used in a crossover design. A portable dynamometer was used to evaluate mechanical nociceptive thresholds at different points on the dorsal laminae, bulbs of the heel, coronary band and sole before and after the horses underwent perineural injection of PDNs or DBs with a local anaesthetic solution (treated group) or an isotonic saline solution (control group). Cornified tissue was removed from the sole and the dorsal aspect of the hoof wall before evaluations of mechanical nociceptive thresholds. Anaesthetising PDNs distal to the DBs increased mechanical nociceptive thresholds compared to baseline values (P <0.001) at sites assessed in the dorsal laminae, sole, and the bulbs of the heels. Anaesthetising DBs increased mechanical nociceptive thresholds compared to baseline values (P <0.01) only at sites assessed at the most proximal aspect of the foot (i.e., coronary band sites). In conclusion, PDNs, not DBs, are primarily responsible for pain signal transmission evoked by pressure in the dorsal laminae of the foot of clinically normal horses.

Mitogen-activated kinase pathway activation in epidermal lamellae in the acute stages of carbohydrate overload laminitis models and the effect of regional deep hypothermia on signalling pathways.

REASONS FOR PERFORMING STUDY: In sepsis models, mitogen-activated protein kinases (MAPKs) are reported to incite inflammatory injury to tissues and are purported to be a therapeutic target. OBJECTIVES: To assess MAPK signalling in lamellae in sepsis-related laminitis (SRL) at different time points after induction of laminitis via carbohydrate overload, and to determine the effect of regional deep hypothermia (RDH) on MAPK signalling. STUDY DESIGN: In vitro study using archived tissue samples. METHODS: Lamellar concentrations of MAPKs were assessed in archived lamellar samples from 2 studies: 1) the starch gruel model of SRL with 3 groups (n = 6/group) of horses (control, onset of fever [DEV] Obel Grade 1 lameness [OG1]); and 2) from limbs maintained at ambient (AMB) and hypothermic (ICE) temperatures (n = 6/group) in animals given a bolus of oligofructose. Immunoblotting and immunolocalisation were used to assess lamellar concentrations and cellular localisation of total and activated (phosphorylated) forms of p38 MAPK, extracellular-regulated kinase (ERK) 1/2, and stress-activated protein kinase/c-jun N terminal kinase (SAPK/JNK) 1/2. RESULTS: Lamellar samples had statistically significant increased concentrations of activated ERK 1/2 at the onset of OG1 laminitis (vs. control) in the starch gruel model, but showed no significant change between ICE and AMB limbs in the RDH model. Phospho-SAPK/JNK 1/2 exhibited a similar significant increase in the OG1 samples, but was also increased in ICE (vs. AMB) limbs. No statistically significant changes in lamellar p38 MAPK concentrations were noted. CONCLUSIONS: Increased concentrations of activated ERK 1/2 and SAPK/JNK in the acute stages of SRL indicate a possible role of these signalling proteins in lamellar injury. Signalling related to ERK 1/2 and SAPK/JNK 1/2 pathways should be further investigated to determine if these play a detrimental role in laminitis and may be therapeutic targets to be manipulated independently of RDH.

Equine insulin receptor and insulin-like growth factor-1 receptor expression in digital lamellar tissue and insulin target tissues.

REASONS FOR PERFORMING STUDY: Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known. OBJECTIVES: The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horses and examine the effect of dietary nonstructural carbohydrate (NSC) on mRNA expression of these receptors in LT, skeletal muscle, liver and two adipose tissue (AT) depots of lean and obese ponies. STUDY DESIGN: In vivo experiment. METHODS: Lamellar tissue samples were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for receptor mRNA expression (n = 8) and immunoblotting for protein expression (n = 3). Archived LT, skeletal muscle, liver and AT from lean and obese mixed-breed ponies fed either a low (~7% NSC as dry matter; 5 lean, 5 obese) or high NSC diet (~42% NSC as dry matter; 6 lean, 6 obese) for 7 days were evaluated by RT-qPCR to determine the effect of body condition and diet on expression of the receptors in different tissues. Significance was set at P≤0.05. RESULTS: Lamellar tissue expresses both InsR isoforms, IGF-1R and Hybrid. LT IGF-1R gene expression was greater than either InsR isoform and InsR-A expression was greater than InsR-B (P≤0.05). Obesity significantly lowered IGF-1R, InsR-A and InsR-B mRNA expression in LT and InsR-A in tailhead AT. High NSC diet lowered expression of all three receptor types in liver; IGF-1R and InsR-A in LT and InsR-A in tailhead AT. CONCLUSIONS: Lamellar tissue expresses IGF-1R, InsR isoforms and Hybrids. The functional characteristics of these receptors and their role in endocrinopathic laminitis warrants further investigation.

Quantitative assessment of the equine hoof using digital radiography and magnetic resonance imaging.

REASONS FOR PERFORMING STUDY: Evaluation of laminitis cases relies on radiographic measurements of the equine foot. Reference values have not been established for all layers of the foot. OBJECTIVES: To establish normal hoof wall and sole measurements using digital radiography (DR) and magnetic resonance imaging (MRI) and to document tissue components present in the dorsal hoof wall and solar layers seen on DR. STUDY DESIGN: Prospective observational case-control study. METHODS: Digital radiography and MRI were performed on 50 cadaver front feet from 25 horses subjected to euthanasia for nonlameness-related reasons. Four observers measured hoof wall (dorsal, lateral and medial) and sole thickness (sagittal, lateral and medial) using DR and magnetic resonance images. One observer repeated the measurements 3 times. Inter- and intraobserver correlation was assessed. RESULTS: Digital radiography and MRI measurements for the normal hoof wall and sole were established. Inter- and intraobserver pairwise Pearson's correlation for DR (r>0.98) and MRI measurements (r>0.99) was excellent. Based on MRI, the less radiopaque layer on DR is comprised of the stratum lamellatum and stratum reticulare. CONCLUSIONS: Normal DR and MRI measurements for the hoof wall and sole were established. On DR images, the less radiopaque layer of the foot observed corresponds to the critical tissues injured in laminitis, the strata lamellatum and reticulare. These reference measurements may be used by the clinician to detect soft-tissue changes in the laminitic equine foot and provide a foundation for future research determining changes in these measurements in horses with laminitis.

Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis.

REASONS FOR PERFORMING STUDY: Dysadhesion of laminar basal epithelial cells (LBECs) from the underlying dermis is the central event leading to structural failure in equine laminitis. Although many studies of sepsis-related laminitis have reported multiple events occurring throughout the lamellar tissue, there is minimal information regarding signalling events occurring specifically in LBECs. OBJECTIVES: To determine signalling events in the LBECs during the early stages of carbohydrate-induced laminitis. STUDY DESIGN: Experimental study. METHODS: Eight horses were given an overload of carbohydrate (CHO) consisting of corn starch mixture via nasogastric tube. Prior to administration of CHO, lamellar biopsies were taken from the left forefoot (control [CON]). Biopsies were taken from the left hind foot at the onset of fever (developmental [DEV]) and from the right forefoot at the onset of Obel grade 1 lameness (OG1). Laminar basal epithelial cells were isolated from cryosections using a laser capture microdissection (LCM) microscope. Next generation sequencing (RNA-seq) was used to identify transcripts expressed in the LBECs for each time point and bioinformatic analysis was performed with thresholds for between group comparisons set at a greater than 2-fold change and P value ≤0.05. RESULTS: Forty genes (22 increased/18 decreased) were significantly different from DEV time vs. CON and 107 genes (57 increased/50 decreased) were significantly different from OG1 time vs. CON. Significant increases in inflammatory genes were present in addition to significantly altered expression of genes related to extracellular matrix composition, stability and turnover. CONCLUSIONS: Signalling related to inflammatory response and extracellular matrix regulation was strongly represented at the DEV and OG1 times. These results indicate that the LBEC is not only a casualty but also an active participant in lamellar events leading to structural failure of the digital lamellae in equine laminitis.

Laminar inflammatory events in lean and obese ponies subjected to high carbohydrate feeding: Implications for pasture-associated laminitis.

REASONS FOR PERFORMING STUDY: Acute, massive enteral carbohydrate overload is associated with laminar inflammation in equids; it is unclear if the same is true for a more prolonged period of moderate dietary carbohydrate intake. OBJECTIVES: To characterise laminar inflammation in ponies exposed to a dietary carbohydrate challenge meant to mimic acute pasture exposure. STUDY DESIGN: In vivo experiment. METHODS: Mixed-breed ponies (n = 22) received a diet of hay chop (nonstructural carbohydrate [NSC] ∼7% on a dry matter [DM] basis) for 4 weeks prior to initiation of the experimental feeding protocol. Following dietary acclimation, ponies were stratified into either Lean (n = 11, body condition score [BCS] ≤4) or Obese (n = 11, BCS ≥7) groups and each group further stratified to either remain on the control, low NSC diet (n = 5 each for Obese and Lean) or receive a high NSC diet (hay chop supplemented with sweet feed and oligofructose, total diet ∼42% NSC; n = 6 each for Obese and Lean) for a period of 7 days. Laminar samples were collected following euthanasia and sections stained immunohistochemically for CD163, MAC387/calprotectin and cyclo-oxygenase-2 (COX-2) using commercially available antibodies. The number of CD163 (+) and MAC387(+) cells was quantified for each section; the distribution of COX-2 expression was qualitatively assessed. Laminar mRNA concentrations of several proinflammatory molecules (interleukin-1ß [IL-1ß], IL-6, tumour necrosis factor-α [TNFα], IL-8, IL-10, monocyte chemoattractant protein-1 [MCP-1], MCP-2), inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), E-selectin, plasminogen activator inhibitor-1 (PAI-1) and COX-2 were evaluated using real-time quantitative polymerase chain reaction (qPCR). RESULTS: High carbohydrate feeding resulted in no increase in laminar proinflammatory cytokine expression; laminar COX-2 expression was increased by high carbohydrate feeding. No laminar leucocyte infiltration was observed in response to high carbohydrate feeding. CONCLUSIONS: These results suggest that the marked laminar inflammation observed in models of sepsis-associated laminitis may not play a central role in the pathophysiology of pasture-associated laminitis.

Effect of dietary nonstructural carbohydrate content on activation of 5'-adenosine monophosphate-activated protein kinase in liver, skeletal muscle, and digital laminae of lean and obese ponies.

BACKGROUND: In EMS-associated laminitis, laminar failure may occur in response to energy failure related to insulin resistance (IR) or to the effect of hyperinsulinemia on laminar tissue. 5'-Adenosine-monophosphate-activated protein kinase (AMPK) is a marker of tissue energy deprivation, which may occur in IR. HYPOTHESIS/OBJECTIVES: To characterize tissue AMPK regulation in ponies subjected to a dietary carbohydrate (CHO) challenge. ANIMALS: Twenty-two mixed-breed ponies. METHODS: Immunohistochemistry and immunoblotting for total AMPK and phospho(P)-AMPK and RT-qPCR for AMPK-responsive genes were performed on laminar, liver, and skeletal muscle samples collected after a 7-day feeding protocol in which ponies stratified on body condition score (BCS; obese or lean) were fed either a low-CHO diet (ESC + starch, approximately 7% DM; n = 5 obese, 5 lean) or a high-CHO diet (ESC + starch, approximately 42% DM; n = 6 obese, 6 lean). RESULTS: 5'-Adenosine-monophosphate-activated protein kinase was immunolocalized to laminar keratinocytes, dermal constituents, and hepatocytes. A high-CHO diet resulted in significantly decreased laminar [P-AMPK] in lean ponies (P = .03), but no changes in skeletal muscle (lean, P = .33; obese, P = .43) or liver (lean, P = .84; obese, P = .13) [P-AMPK]. An inverse correlation existed between [blood glucose] and laminar [P-AMPK] in obese ponies on a high-CHO diet. CONCLUSIONS AND CLINICAL IMPORTANCE: Laminar tissue exhibited a normal response to a high-CHO diet (decreased [P-AMPK]), whereas this response was not observed in liver and skeletal muscle in both lean (skeletal muscle, P = .33; liver, P = .84) and obese (skeletal muscle, P = .43; liver, P = .13) ponies.

Expression and activity of collagenases in the digital laminae of horses with carbohydrate overload-induced acute laminitis.

BACKGROUND: Matrix metalloproteinases (MMP) are hypothesized to degrade structurally important components of the laminar extracellular matrix (ECM) in horses with laminitis. OBJECTIVE: To compare levels of expression of stromelysin-1 (MMP-3), collagenases (MMP-1, -13), and membrane type-MMPs (MMP-14, -15, -16), and the distribution of their ECM substrates, in laminae of healthy horses and horses with carbohydrate overload laminitis. ANIMALS: Twenty-five adult horses. METHODS: Gene and protein expression were determined in extracts of laminae using real-time quantitative polymerase chain reaction and Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis. Distribution of MMP-13 and ECM components was determined using indirect immunofluorescent microscopy of nonfixed frozen sections. ECM morphology was assessed by hematoxylin and eosin staining. RESULTS: Of the genes studied, only those encoding MMP-1 and -13 were upregulated in CHO-induced laminitis; MMP-1 at Obel grade (OG)1 lameness and MMP-13 at OG3 lameness. Laminar MMP-1 was present as 52 kDa proenzyme only. MMP-13 was present as pro- (61 kDa) and processed (48 kDa) enzyme. MMP-13 localized to the basal epithelium of the secondary epidermal laminae and its increased expression were accompanied by the appearance in secondary dermal laminae (SDL) of multiple foci that were devoid of collagen I, fibronectin, chondroitin and keratan sulfate glycosaminoglycans, and eosin-staining material. CONCLUSIONS AND CLINICAL RELEVANCE: MMP-13 is upregulated in laminae of horses with CHO-induced OG3 lameness and, by degrading components of the ECM, may contribute to the formation of ECM-free lesions (gaps or tears) that appear in the SDL with OG3 lameness.

Regulation of hypoxia-inducible factor-1α and related genes in equine digital lamellae and in cultured keratinocytes.

REASONS FOR PERFORMING STUDY: Hypoxia-inducible factor-1α (HIF-1A) is an important protein in the regulation/induction of many genes in the cellular and tissue response to hypoxia and a central mediator in inflammatory signalling. As both hypoxia and inflammatory events are purported to occur in the lamellar epidermis in sepsis-related laminitis in the equid, HIF-1A may play a central role in this disease process. OBJECTIVESS: To assess the regulation of HIF-1A and HIF-1A-related genes in the equine keratinocyte in vitro and in the lamellar tissue of horses with sepsis-related laminitis. STUDY DESIGN: In vivo and in vitro experiments. METHODS: Real-time quantitative PCR (RT-qPCR) and immunoblotting were performed to assess the mRNA and protein concentrations of HIF-1A and the mRNA concentrations of HIF-1A-related genes in cultured equine keratinocytes and in lamellar samples from black walnut extract (BWE)- and carbohydrate overload (CHO)-induced laminitis. Hypoxia-inducible factor-1α was further localised via indirect immunofluorescence in frozen lamellar tissue sections. RESULTS: Hypoxia-inducible factor-1α appears to be regulated primarily at the post transcriptional level in the cultured equine keratinocyte, resulting in increased HIF-1A in response to hypoxia but not to lipopolysaccharide exposure. Hypoxia-inducible factor-1α is present at high concentrations in the normal equine lamina, and is increased in Obel grade 1 (OG1) stage laminitis in the CHO model of laminitis. Equine lamellar mRNA concentrations of cyclo-oxygenase-2 and inducible nitric oxide synthase, but not glucose transporter 1, are increased in the BWE and CHO models of laminitis. CONCLUSIONS AND POTENTIAL RELEVANCE: These data indicate that the normal equine lamellae are profoundly hypoxic in comparison with other tissues. The increased mRNA concentrations of cyclo-oxygenase-2 and inducible nitric oxide synthase 2 in equine keratinocytes exposed to hypoxia and lipopolysaccharide, and in lamellar tissue from BWE and CHO models of sepsis-related laminitis, suggest that the marked lamellar inflammatory gene expression in sepsis-related laminitis may be due to an interaction of constitutively high lamellar keratinocyte HIF-1A signalling with inflammatory signalling, possibly induced by circulating inflammatory mediators.

Continuous digital hypothermia initiated after the onset of lameness prevents lamellar failure in the oligofructose laminitis model.

REASONS FOR PERFORMING STUDY: Prophylactic digital hypothermia reduces the severity of acute laminitis experimentally but there is no evidence for its efficacy as a treatment once lameness has already developed. OBJECTIVES: To investigate the therapeutic effects of digital hypothermia, applied after the onset of lameness, in an experimental acute laminitis model. STUDY DESIGN: Randomised, controlled (within subject), blinded, experimental trial. METHODS: Eight Standardbred horses underwent laminitis induction using the oligofructose model. Once lameness was detected at the walk, one forelimb was continuously cooled (CRYO), with the other forelimb maintained at ambient temperature (NON-RX). Dorsal lamellar sections (proximal, middle and distal) harvested 36 h after the onset of lameness/initiation of cryotherapy were analysed by 2 blinded observers: laminitis pathology was scored (0 [normal] to 4 [severe]) and morphometric analyses performed. RESULTS: Median (interquartile range) histological scores were greater (P<0.05) in NON-RX (proximal 2.8 [2.5-4]; middle 3.5 [2-4]; distal 2.5 [2-3.8]) compared with CRYO limbs (proximal 0.5 [0.5-1.4]; middle 1 [0.6-1]; distal 0.75 [0.5-1]). There was complete physical separation of lamellar dermis from epidermis (score of 4) in 4 of the NON-RX feet at one or more section level(s), which was not observed in any CRYO sections. Histomorphometry was thus limited to sections that remained intact; there was a trend of increased total (TELL) and secondary (SELL) epidermal lamellar length and decreased secondary epidermal lamellar width (SELW) in NON-RX limbs compared with CRYO at all 3 levels; differences were significant (P<0.05) for SELL and SELW in the distal sections. CONCLUSIONS: Digital hypothermia reduced the severity of lamellar injury and prevented lamellar structural failure (complete dermoepidermal separation) when initiated at the detection of lameness in an acute laminitis model. This study provides the first evidence to support the use of therapeutic digital hypothermia as a treatment for acute laminitis.

Progress in a replicated selection for elevated blood ethanol concentrations in HDID mice.

Drinking in the dark (DID) is a limited access ethanol-drinking phenotype in mice. High Drinking in the Dark (HDID-1) mice have been bred for 27 selected generations (S27) for elevated blood ethanol concentrations (BECs) after a 4-h period of access to 20% ethanol. A second replicate line (HDID-2) was started later from the same founder population and is currently in S20. An initial report of response to selection in HDID-1 was published after S11. This article reports genetic and behavioral characteristics of both lines in comparison with the HS controls. Heritability is low in both replicates (h(2) = 0.09) but the lines have shown 4-5 fold increases in BEC since S0; 80% of HDID-1 and 60% of HDID-2 mice reach BECs greater than 1.0 mg/ml. Several hours after a DID test, HDID mice show mild signs of withdrawal. Although not considered during selection, intake of ethanol (g/kg) during the DID test increased by approximately 80% in HDID-1 and 60% in HDID-2. Common genetic influences were more important than environmental influences in determining the similarity between BEC and intake for HDID mice. Analysis of the partitioning of intake showed that 60% of intake is concentrated in the last 2 h of the 4 h session. However, this has not changed during selection. Hourly BECs during the DID test reach peak levels after 3 or 4 h of drinking. HDID mice do not differ from HS mice in their rate of elimination of an acute dose of alcohol.

Prophylactic digital cryotherapy is associated with decreased incidence of laminitis in horses diagnosed with colitis.

REASONS FOR PERFORMING THE STUDY: Recent research suggested that prophylactic digital cryotherapy (ICE) improved lameness scores, diminished histological changes and early laminar inflammatory signalling in horses following oligofructose administration. In clinical practice, horses at risk for sepsis-associated laminitis receive ICE. Evidence to support this practice is lacking. OBJECTIVES: To determine factors associated with development of laminitis in horses diagnosed with colitis, including ICE. STUDY DESIGN: Multicentre retrospective case series. METHODS: Medical records for horses admitted to 2 university hospitals diagnosed with colitis with evidence of systemic inflammatory response from 2002 to 2012 were reviewed. Horses were excluded if they exhibited signs of laminitis at admission, were ponies, miniature or draught breeds, or <2 years old. Data were analysed using univariate and multivariate logistic regression. RESULTS: Twenty-seven of 130 horses (21%) developed laminitis. Seven of 69 (10%) horses treated with ICE developed laminitis compared with 20/61 (33%) horses that developed laminitis but did not receive ICE. Factors associated with laminitis included site of hospitalisation, admission respiratory rate (↑) and blood L-lactate (↑), and ICE (↓), P<0.05. Horses treated with ICE had 10 times less odds of developing laminitis compared with horses treated without ICE (odds ratio 0.11, 95% confidence limit 0.03-0.44). Sixteen horses (16/130, 12%) were subjected to euthanasia in hospital. Fourteen of these horses had laminitis and 2 did not develop laminitis. Survival for horses with colitis that developed laminitis was 13/27 (48%) compared with survival for horses with colitis that did not develop laminitis, 101/103 (98%). CONCLUSION: Laminitis occurred in more clinically compromised horses. Use of ICE reduced the incidence of clinical laminitis in the study population suggesting that digital cryotherapy is an effective prophylactic strategy for the prevention of laminitis in horses with colitis.