Digital lamellar inflammatory signaling in an experimental model of equine preferential weight bearing.

BACKGROUND: Supporting limb laminitis (SLL) is a complication of severe orthopedic disease in horses and is often life-limiting, yet the pathophysiology remains obscure. HYPOTHESIS/OBJECTIVES: To investigate the role of digital lamellar inflammatory signaling in the pathophysiology of SLL using a model of unilateral weight bearing, hypothesizing that there would be evidence of lamellar inflammation in limbs subjected to the model. ANIMALS: Thirteen healthy adult Standardbred horses were used for this study (11 geldings, 2 mares; mean age 6.5 ± 2.5 years; mean body weight 458.3 ± 32.8 kg). METHODS: Randomized controlled experimental study. A steel shoe with a custom insert was applied to a randomly selected front foot of 7 horses; 6 horses were unshod and served as controls. After 92 hours, all horses were humanely euthanized, and digital lamellar samples were collected. Lamellar protein and mRNA were isolated and used to perform western blot and PCR. RESULTS: Lamellar concentrations of IL-6 mRNA were higher in SL tissue than IL HIND tissue (median [25%-75%] normalized copy number 191 [111-3060] and 48 [25-74], respectively; P=.003), and lamellar concentrations of COX-2 mRNA were higher in SL tissue than CON tissue (normalized copy number 400 [168-634] and 125 [74-178], respectively; P=.007). Lamellar concentrations of IL-1B, IL-10, and COX-1 mRNA were not significantly different between groups. The concentrations of phosphorylated (activated) STAT1 and STAT3 proteins were higher in SL (0.5 [0.35-0.87] and 1.35 [1.1-1.7], respectively) compared to CON (0.24 [0.09-0.37] and 0.31 [0.16-037]) and UL HIND (0.27 [0.19-0.37] and 0.38 [0.24-0.5]); P=0.01 and P<0.001. CONCLUSIONS AND CLINICAL IMPORTANCE: Lamellar inflammatory signaling was higher in tissue from horses subjected to prolonged unilateral weight-bearing, suggesting that these pathways could be relevant to the pathophysiology of SLL.

Diagnostic evaluation of insulin and glucose dynamics in light-breed horses receiving dexamethasone.

Objective: Insulin dysregulation is a hallmark of equine metabolic syndrome (EMS) and increases the risk for development of laminitis. Accurate diagnosis of insulin dysregulation is crucial for implementation of preventative strategies in this population. The objective was to assess the effects of dexamethasone administration on insulin and glucose dynamics in light-breed horses and assess the agreement of various diagnostic tests for insulin dysregulation [basal [insulin] (BI), oral sugar test (OST), and combined glucose-insulin test (CGIT)]. Animal: Fourteen adult light-breed horses. Procedure: Prospective, experimental study to assess insulin and glucose dynamics by performing basal insulin, OST, and CGIT before (baseline) and post-dexamethasone administration (0.08 mg/kg, PO, q24h) for 7 d. Insulin and glucose dynamics were assessed by the BI, OST, CGIT, and insulin sensitivity proxy measurements (RISQI, QUICKI, FGIR, HOMA-IR, IG) at the baseline and post-dexamethasone time points. Results: The OST area under the insulin and glucose curves were increased following dexamethasone treatment (P < 0.001 and P < 0.01, respectively). Basal insulin, OST [insulin] at 60 min and CGIT [insulin] at 45 min were increased at the post-dexamethasone time point (P < 0.001, < 0.001, and < 0.01). Similarly, time spent in the positive glucose phase during the CGIT was longer at the post-dexamethasone time point (P < 0.001). The proxy measurements for insulin sensitivity (RISQI, QUICKI, FGIR) were decreased (P < 0.01) and the proxy measurements for insulin resistance (HOMA-IR) and ß-cell function (IG) were increased after dexamethasone administration (P < 0.01). More horses were classified with following dexamethasone administration, based on the diagnostic criteria for basal insulin (P = 0.03), OST (P = 0.01), and CGIT (P < 0.01). Kappa coefficients, measuring agreement between basal insulin, OST, and CGIT, showed none to moderate agreement at the baseline time point. Conclusion: Dexamethasone administration at 0.08 mg/kg, PO, q24h for 7 d worsened insulin dysregulation in adult light-breed horses based on findings of a basal insulin, OST, CGIT, and insulin sensitivity proxy measurements. There was none to moderate agreement between the basal insulin, OST, CGIT for the diagnosis of insulin dysregulation. Clinical relevance: Horses administered dexamethasone at a dose of 0.08 mg/kg, PO, q24h for 7 d should be considered insulin dysregulation and appropriate preventative strategies should be implemented. The variability of diagnostic performance of common tests for insulin dysregulation (basal insulin, OST, CGIT) may affect clinical decisions; therefore, performing multiple tests, including proxy measurements, may improve diagnostic accuracy of insulin dysregulation.

Objectif: La dysrégulation de l'insuline est une caractéristique du syndrome métabolique équin (EMS) et augmente le risque de développement de la fourbure. Un diagnostic précis de la dysrégulation de l'insuline est crucial pour la mise en oeuvre de stratégies préventives dans cette population. L'objectif était d'évaluer les effets de l'administration de dexaméthasone sur la dynamique de l'insuline et du glucose chez les chevaux de race légère et d'évaluer la concordance de divers tests de diagnostic pour le dérèglement de l'insuline [insuline basale] (BI), test de sucre oral (OST) et un test glucose-insuline combiné (CGIT). Animal: Quatorze chevaux adultes de race légère. Procédure: Étude prospective et expérimentale pour évaluer la dynamique de l'insuline et du glucose en effectuant l'insuline basale, l'OST et le CGIT avant (valeur de base) et après l'administration de dexaméthasone (0,08 mg/kg, PO, q24h) pendant 7 jours. La dynamique de l'insuline et du glucose a été évaluée par les mesures indirectes de BI, de l'OST, du CGIT et de la sensibilité à l'insuline (RISQI, QUICKI, FGIR, HOMA-IR, IG) aux points temporels de base et post-dexaméthasone. Résultats: La zone OST sous les courbes d'insuline et de glucose a augmenté après le traitement à la dexaméthasone (P < 0,001 et P < 0,01, respectivement). L'insuline basale, l'OST [insuline] à 60 minutes et le CGIT [insuline] à 45 minutes ont augmenté au point temporel post-dexaméthasone (P < 0,001, < 0,001 et < 0,01). De même, le temps passé dans la phase de glucose positif pendant le CGIT était plus long au moment post-dexaméthasone (P < 0,001). Les mesures indirectes de la sensibilité à l'insuline (RISQI, QUICKI, FGIR) ont diminué (P < 0,01) et les mesures indirectes de la résistance à l'insuline (HOMA-IR) et de la fonction des cellules ß (IG) ont augmenté après l'administration de dexaméthasone (P < 0,01). Plus de chevaux ont été classés avec l'administration suivante de dexaméthasone, sur la base des critères de diagnostic de l'insuline basale (P = 0,03), OST (P = 0,01) et CGIT (P < 0,01). Les coefficients Kappa, mesurant la concordance entre l'insuline basale, l'OST et le CGIT, ont montré une concordance nulle à modérée au point de référence. Conclusion: L'administration de dexaméthasone à 0,08 mg/kg, PO, toutes les 24 h pendant 7 jours a aggravé la dysrégulation de l'insuline chez les chevaux adultes de race légère d'après les résultats d'une insuline basale, d'OST, de CGIT et de mesures indirectes de la sensibilité à l'insuline. Il n'y avait aucun accord à modéré entre l'insuline basale, l'OST, le CGIT pour le diagnostic de dysrégulation de l'insuline. Pertinence clinique: Les chevaux ayant reçu de la dexaméthasone à une dose de 0,08 mg/kg, PO, q24h pendant 7 jours doivent être considérés comme ayant un dérèglement de l'insuline et des stratégies préventives appropriées doivent être mises en oeuvre. La variabilité des performances diagnostiques des tests courants de dysrégulation de l'insuline (insuline basale, OST, CGIT) peut affecter les décisions cliniques; par conséquent, la réalisation de plusieurs tests, y compris des mesures indirectes, peut améliorer la précision du diagnostic du dérèglement de l'insuline.(Traduit par Dr Serge Messier).

Plasma amino acid concentrations during experimental hyperinsulinemia in 2 laminitis models.

BACKGROUND: Endocrinopathic laminitis develops in association with insulin dysregulation, but the role of insulin in the pathogenesis remains unclear. Hyperinsulinemia can cause hypoaminoacidemia, which is associated with integumentary lesions in other species and therefore warrants investigation as a potential mechanism in laminitis. OBJECTIVE: Evaluate plasma amino acid concentrations in the euglycemic-hyperinsulinemic clamp (EHC) and prolonged glucose infusion (PGI) laminitis models. ANIMALS: Sixteen Standardbred horses. METHODS: Prospective experimental study. Plasma amino acid concentrations were measured in samples collected every 6 hours from horses that underwent a 48-hour EHC (n = 8) or 66-hour PGI (n = 8) after a 24- or 6-hour baseline period in EHC and PGI groups, respectively. RESULTS: Fifteen of the 20 measured amino acid concentrations decreased over time in both EHC and PGI horses (P < 0.001). The median percentage change from baseline for these amino acids was: histidine (EHC: 41.5%; PGI: 43.9%), glutamine (EHC: 51.8%; PGI: 35.3%), arginine (EHC: 51.4%; PGI: 41%), glutamic acid (EHC: 52.4%; PGI: 31.7%), threonine (EHC: 62.8%; PGI: 25.2%), alanine (EHC: 48.9%; PGI: 19.5%), proline (EHC: 56.2%; PGI: 30.3%), cystine (EHC: 34.9%; PGI: 31.2%), lysine (EHC: 46.4%; PGI: 27.8%), tyrosine (EHC: 27.5%; PGI: 16.9%), methionine (EHC: 69.3%; PGI: 50.8%), valine (EHC: 50.8%; PGI: 34.4%), isoleucine (EHC: 60.8%; PGI: 38.7%), leucine (EHC: 48.2%; PGI: 36.6%), and phenylalanine (EHC: 16.6%; PGI: 12.1%). CONCLUSIONS AND CLINICAL IMPORTANCE: Hypoaminoacidemia develops in EHC and PGI laminitis models. The role of hypoaminoacidemia in the development of hyperinsulinemia-associated laminitis warrants further investigation.

Lamellar perfusion and energy metabolism in a preferential weight bearing model.

BACKGROUND: Supporting limb laminitis (SLL) is suspected to be caused by lamellar ischaemia as a consequence of increased mechanical load. OBJECTIVES: Examine the effects of prolonged preferential weight bearing (PWB) on lamellar perfusion and metabolism. STUDY DESIGN: In vivo experiment. METHODS: Microdialysis probes were inserted in the lamellar and sublamellar dermis of one forelimb in 13 Standardbred horses. In six horses, a platform shoe (contralateral forelimb) was used to induce increased load on the microdialysis-instrumented forelimb (PWB). The remaining seven horses were controls (CON). All horses were housed in stocks with limb weight distribution logged continuously for 92 hours. Microdialysate was collected and analysed every 4 hours for glucose, lactate, pyruvate, and lactate to pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar perfusion. Data were analysed using a mixed-effects linear regression model. RESULTS: Median [IQR] load on the microdialysis-instrumented limb was equivalent to 38.7% bwt. [37.3-40.3] in PWB and 27.3% bwt. [26.6-28] in CON. Limb offloading frequency increased in CON (P < .001) but not PWB (P = .2). Lamellar microdialysate glucose decreased in PWB (P < .001) and CON (P = .004), however, the rate of decrease was higher in PWB (P = .007). Lamellar L:P increased in PWB (P < .001) and peaked at 196 [79-656], whereas L:P did not change over time in CON (P = .6) and peaked at 42 [41-49]. Lamellar urea clearance decreased in PWB (P < .001) but not in CON (P = .3). Sublamellar L:P and urea clearance did not change over time in either group (P > .05). MAIN LIMITATIONS: The PWB model may not be representative of naturally occurring SLL. CONCLUSIONS: Evidence of lamellar ischaemia (increased L:P and decreased urea clearance) was detected exclusively in the lamellar dermis of PWB feet subjected to persistently increased load. Lamellar ischaemia is a consequence of increased mechanical load and likely contributes to the development of SLL.

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

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-1ß, 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-1ß (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.

Intrasinus bolstering of traumatic maxillary sinus fractures by using Foley catheter balloons in two foals.

OBJECTIVE: To describe the repair of unstable facial fractures by using Foley catheter balloons as intrasinus bolsters. STUDY DESIGN: Case report ANIMALS: Two weanling foals with unilateral fractures of the sinus and orbit secondary to kick injuries. Preoperative imaging that included positive contrast dacrocystorhinography and computed tomography confirmed severe comminution of facial fractures and nasolacrimal duct disruption in both foals. METHODS: Small bone fragments were surgically removed, and large fragments were retained even when denuded of periosteum. Repair procedures included nasolacrimal canaliculosinusotomy and suturing fracture fragments together with polydioxanone sutures. After fixation, the fracture fragments could be depressed into the sinus with manual pressure, so two intrasinus Foley catheters were placed to bolster the sinus wall, with the tubing exiting through a frontal sinus trephine. The skin was completely closed over the fractures. Catheters and nasolacrimal stenting were maintained in place during fracture healing. RESULTS: One foal prematurely dislodged catheters and nasolacrimal stent 11 days after fixation. The catheters and stenting were removed as planned 4 weeks after surgery in the second foal. Wound, fracture healing, and overall cosmesis was good in both foals, and epiphora resolved. Surgical site infection, sinusitis, and sequestration did not occur. Both foals became high-level performance horses with acceptable cosmetic outcome and good bilateral nasal airflow. CONCLUSION: Foley catheter balloons supported sinus fracture repair and maintained stability of the surgical reconstruction during convalescence. CLINICAL SIGNIFICANCE: Suture repair of comminuted sinus fractures can be supported by using Foley catheters, which are readily available.

Lamellar energy metabolism and perfusion in the euglycaemic hyperinsulinaemic clamp model of equine laminitis.

BACKGROUND: Hyperinsulinaemia is associated with the development of endocrinopathic laminitis; however, the mechanisms remain unclear. OBJECTIVES: Evaluate the effects of hyperinsulinaemia on lamellar energy metabolism and perfusion during laminitis development. STUDY DESIGN: In vivo experiment. METHODS: Eight Standardbred horses were instrumented with a microdialysis probe in the lamellae of a forelimb. A 24 hours baseline period (BASELINE) was followed by 48 hours of a continuous euglycaemic hyperinsulinaemic clamp (EHC) from 24 to 72 hours (CLAMP). Microdialysate was collected every 6 hours and analysed for glucose, lactate and pyruvate concentrations and lactate-to-pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar tissue perfusion. Archived microdialysis samples from six identically instrumented Standardbred horses served as controls (CON). Variables were compared over time and between EHC and CON horses using a mixed-effects linear regression model. RESULTS: Glucose concentration decreased during the CLAMP period in CON and EHC horses (P < .001), but there was no difference between CON and EHC (P > .9). Lactate concentration increased during the CLAMP period in CON and EHC horses (P < .001), however, the rate of increase was significantly higher in EHC horses relative to CON (P = .014). There was a relative increase in pyruvate concentration in EHC horses compared with CON during the CLAMP period (P = .03). L:P increased significantly in CON horses during the CLAMP period (P < .001) but not in EHC (P = .1). Urea clearance did not change in CON (P = .9) or EHC (P = .05) during the CLAMP, but did increase in EHC relative to CON (P = .02). MAIN LIMITATIONS: The effects of microdialysis probe implantation on perfusion and metabolism remain unclear. The EHC model may not mimic natural endocrinopathic laminitis. CONCLUSIONS: Laminitis developed without evidence of lamellar hypoperfusion or energy stress. Therapies to improve perfusion are unlikely to affect the initial development of endocrinopathic laminitis.

The effect of continuous digital hypothermia on lamellar energy metabolism and perfusion during laminitis development in two experimental models.

BACKGROUND: Continuous digital hypothermia (CDH) prevents lamellar failure in the euglycaemic hyperinsulinaemic clamp (EHC) and oligofructose (OF) laminitis models, but the mechanisms remain unclear. OBJECTIVES: To evaluate the effects of CDH on lamellar energy metabolism and perfusion in healthy horses and during EHC and OF laminitis models. STUDY DESIGN: In vivo experiment. METHODS: Archived samples were used from Standardbred geldings that received no treatment (CON) (n = 8) or underwent EHC (n = 8) or OF (n = 6) laminitis models. Both forelimbs were instrumented with a lamellar microdialysis system, and one forelimb was cooled (CDH) with the other maintained at ambient temperature (AMB). Microdialysate was collected every 6 hours and analysed for glucose, lactate and pyruvate concentrations and lactate to pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar tissue perfusion. Data were analysed using a mixed-effects linear regression model. RESULTS: Glucose did not change in CDH limbs relative to AMB in CON (P = .3), EHC (P = .3) or OF (P = .6) groups. There was a decrease in lactate (P < .001) and pyruvate (P < .01) in CDH limbs relative to AMB in all groups. L:P decreased in CON CDH relative to CON AMB (P < .001) but was not different in EHC (P = .6) and OF (P = .07) groups. Urea clearance decreased in CDH limbs relative to AMB in CON (P = .002) and EHC (P < .001), but not in OF (P = .4). MAIN LIMITATIONS: The EHC model may not mimic natural endocrinopathic laminitis. CONCLUSIONS: CDH caused a marked decrease in lamellar glucose metabolism (CON, EHC and OF) and perfusion (CON and EHC) without affecting lamellar glucose concentration. Although cellular energy failure is not a primary pathophysiological event in EHC and OF laminitis models, CDH may act by limiting energy supply to pathologic cellular processes whilst preserving those critical to lamellar homoeostasis.

Association of sustained supraphysiologic hyperinsulinemia and inflammatory signaling within the digital lamellae in light-breed horses.

BACKGROUND: Hyperinsulinemia is associated with equine laminitis, and digital lamellar inflammation in equine metabolic syndrome-associated laminitis (EMSAL) is modest when compared with sepsis-associated laminitis. OBJECTIVES: To characterize digital lamellar inflammation in horses in a euglycemic-hyperinsulinemic clamp (EHC) model of laminitis. ANIMALS: Sixteen healthy adult Standardbred horses. METHODS: Prospective experimental study. Horses underwent EHC or saline infusion (CON) for 48 hours or until the onset of Obel grade 1 laminitis. Horses were euthanized, and digital lamellar tissue was collected and analyzed via polymerase chain reaction (pro-inflammatory cytokine and chemokine genes-CXCL1, CXCL6, CXCL8, IL-6, MCP-1, MCP-2, IL-1ß, IL11, cyclooxygenases 1 and 2, tumor necrosis factor alpha [TNF-α], E-selectin, and ICAM-1), immunoblotting (phosphorylated and total signal transducer and activator of transcription 1 [STAT1], STAT3, and p38MAPK), and immunohistochemistry (markers of leukocyte infiltration: CD163, MAC387). RESULTS: Lamellar mRNA concentrations of IL-1ß, IL-6, IL-11, COX-2, and E-selectin were increased; the concentration of COX-1 was decreased; and concentrations of CXCL1, CXCL6, MCP-1, MCP-2, IL-8, TNF-α and ICAM-1 were not significantly different in the EHC group compared to the CON group (P ≤ .003). Lamellar concentrations of phosphorylated STAT proteins (P-STAT1 [S727], P-STAT1 [Y701], P-STAT3 [S727], and P-STAT3 [Y705]) were increased in the EHC group compared to the CON group, with phosphorylated STAT3 localizing to nuclei of lamellar basal epithelial cells. There was no change in the lamellar concentration of P-p38 MAPK (T180/Y182), but the concentration of total p38 MAPK was decreased in the EHC samples. There was no evidence of notable lamellar leukocyte emigration. CONCLUSIONS AND CLINICAL IMPORTANCE: These results establish a role for lamellar inflammatory signaling under conditions associated with EMSAL.

The effect of hypothermia on influx of leukocytes in the digital lamellae of horses with oligofructose-induced laminitis.

Sepsis-related laminitis (SRL) is a common complication in the septic/endotoxemic critically-ill equine patient, in which lamellar injury and failure commonly lead to crippling distal displacement of the distal phalanx. Similar to organ injury in human sepsis, lamellar injury in SRL has been associated with inflammatory events, including the influx of leukocytes into the lamellar tissue and markedly increased expression of a wide array of inflammatory mediators at the onset of Obel grade 1 (OG1) laminitis. The only treatment reported both clinically and experimentally to protect the lamellae in SRL, local hypothermia ("cryotherapy"), has been demonstrated to effectively inhibit lamellar expression of multiple inflammatory mediators when initiated at the time of administration of a carbohydrate overload in experimental models of SRL. However, the effect of hypothermia on leukocyte influx into affected tissue has not been assessed. We hypothesized that cryotherapy inhibits leukocyte emigration into the digital lamellae in SRL. Immunohistochemical staining using leukocyte markers MAC387 (marker of neutrophils, activated monocytes) and CD163 (monocyte/macrophage-specific marker) was performed on archived lamellar tissue samples from an experimental model of SRL in which one forelimb was maintained at ambient temperature (AMB) and one forelimb was immersed in ice water (ICE) immediately following enteral oligofructose administration (10g/kg, n=14 horses). Lamellae were harvested at 24h post-oligofructose administration (DEV, n=7) or at the onset of OG1 laminitis (OG1, n=7). Both MAC387-positive and CD163-positive cells were counted by a single blinded investigator on images [n=10 (40× fields/digit for MAC387 and 20x fields/digit for CD163)] obtained using Aperio microscopy imaging analysis software. Data were assessed for normality and analyzed with a paired t-test and one-way ANOVA with significance set at p<0.05. MAC387-positive cells were present in low numbers in the lamellar tissue and were decreased in the hypothermic limbs (vs. AMB limbs, p<0.05) in the OG1 group; no change in CD163-positive cell numbers was noted across the conditions of the model. This study demonstrated that hypothermia of the distal limbs instituted early in the disease process in the horse at risk of SRL significantly attenuates the increase of MAC387-positive leukocytes in the digital lamellae, but has minimal effect on increases in lamellar concentrations of the major leukocyte cell type present in that tissue, CD163-positive mononuclear cells.

Impact of laminitis on the canonical Wnt signaling pathway in basal epithelial cells of the equine digital laminae.

The digital laminae is a two layer tissue that attaches the distal phalanx to the inner hoof wall, thus suspending the horse's axial skeleton in the hoof capsule. This tissue fails at the epidermal:dermal junction in laminitic horses, causing crippling disease. Basal epithelial cells line the laminar epidermal:dermal junction, undergo physiological change in laminitic horses, and lose versican gene expression. Versican gene expression is purportedly under control of the canonical Wnt signaling pathway and is a trigger for mesenchymal-to-epithelial transition; thus, its repression in laminar epithelial cells of laminitic horses may be associated with suppression of the canonical Wnt signaling pathway and loss of the epithelial cell phenotype. In support of the former contention, we show, using laminae from healthy horses and horses with carbohydrate overload-induced laminitis, quantitative real-time polymerase chain reaction, Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis, and immunofluorescent tissue staining, that positive and negative regulatory components of the canonical Wnt signaling pathway are expressed in laminar basal epithelial cells of healthy horses. Furthermore, expression of positive regulators is suppressed and negative regulators elevated in laminae of laminitic compared to healthy horses. We also show that versican gene expression in the epithelial cells correlates positively with that of ß-catenin and T-cell Factor 4, consistent with regulation by the canonical Wnt signaling pathway. In addition, gene and protein expression of ß-catenin correlates positively with that of integrin ß4 and both are strongly suppressed in laminar basal epithelial cells of laminitic horses, which remain E-cadherin(+)/vimentin(-), excluding mesenchymal transition as contributing to loss of the adherens junction and hemidesmosome components. We propose that suppression of the canonical Wnt signaling pathway, and accompanying reduced expression of ß catenin and integrin ß4 in laminar basal epithelial cells reduces cell:cell and cell:basement membrane attachment, thus, destabilizing the laminar epidermal:dermal junction.

Distribution and processing of a disintegrin and metalloproteinase with thrombospondin motifs-4, aggrecan, versican, and hyaluronan in equine digital laminae.

OBJECTIVE: To determine the expression and distribution of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), its substrates aggrecan and versican, and their binding partner hyaluronan in laminae of healthy horses. SAMPLE: Laminae from the forelimb hooves of 8 healthy horses. PROCEDURES: Real-time quantitative PCR assay was used for gene expression analysis. Hyaluronidase, chondroitinase, and keratanase digestion of lamina extracts combined with SDS-PAGE and western blotting were used for protein and proteoglycan analysis. Immunofluorescent and immunohistochemical staining of tissue sections were used for protein and hyaluronan localization. RESULTS: Genes encoding ADAMTS-4, aggrecan, versican, and hyaluronan synthase II were expressed in laminae. The ADAMTS-4 was predominantly evident as a 51-kDa protein bearing a catalytic site neoepitope indicative of active enzyme and in situ activity, which was confirmed by the presence of aggrecan and versican fragments bearing ADAMTS-4 cleavage neoepitopes in laminar protein extracts. Aggrecan, versican, and hyaluronan were localized to basal epithelial cells within the secondary epidermal laminae. The ADAMTS-4 localized to these cells but was also present in some cells in the dermal laminae. CONCLUSIONS AND CLINICAL RELEVANCE: Within digital laminae, versican exclusively and aggrecan primarily localized within basal epithelial cells and both were constitutively cleaved by ADAMTS-4, which therefore contributed to their turnover. On the basis of known properties of these proteoglycans, it is possible that they can protect the basal epithelial cells of horses from biomechanical and concussive stress.

Effects of cleavage by a disintegrin and metalloproteinase with thrombospondin motifs-4 on gene expression and protein content of versican and aggrecan in the digital laminae of horses with starch gruel-induced laminitis.

OBJECTIVE: To determine whether increased gene expression of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) in laminae of horses with starch gruel-induced laminitis was accompanied by increased enzyme activity and substrate degradation. SAMPLE: Laminae from the forelimb hooves of 8 healthy horses and 17 horses with starch gruel-induced laminitis (6 at onset of fever, 6 at onset of Obel grade 1 lameness, and 5 at onset of Obel grade 3 lameness). PROCEDURES: Gene expression was determined by use of cDNA and real-time quantitative PCR assay. Protein expression and processing were determined via SDS-PAGE and quantitative western blotting. Protein distribution and abundance were determined via quantitative immunofluorescent staining. RESULTS: ADAMTS-4 gene expression was increased and that of versican decreased in laminitic laminae, compared with expression in healthy laminae. Catalytically active ADAMTS-4 also was increased in the tissue, as were ADAMTS-4-cleavage fragments of versican. Immunofluorescent analyses indicated that versican was depleted from the basal epithelia of laminae of horses at onset of Obel grade 3 lameness, compared with results for healthy laminae, and this was accompanied by regional separation of basal epithelial cells from the basement membrane. Aggrecan gene and protein expression were not significantly affected. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in gene and protein expression of ADAMTS-4 and versican in the basal epithelium of laminitic laminae indicated a fundamental change in the physiology of basal epithelial cells. This was accompanied by and may have caused detachment of these cells from the basement membrane.

Laminar chemokine mRNA concentrations in horses with carbohydrate overload-induced laminitis.

Chemokines play a vital role in leukocyte activation and emigration that reportedly plays a central role in laminar injury in equine laminitis. The purpose of this study was to evaluate the pattern of laminar chemokine expression in horses in the classical carbohydrate overload (CHO)-model of laminitis. Laminar samples were obtained 24h following water administration in the control group (CON, n=8), and at the onset of fever (≥ 102°F, 12-22 h post CHO, DEV group, n=8) and at the onset of lameness (20-48 h post CHO, LAM group, n=8) in induced horses. Real time quantitative PCR was performed on all samples in order to determine laminar mRNA concentrations of both CXC chemokines (CXCL1, CXCL6, CXCL8) and CC chemokines (CCL2 [MCP-1], CCL3 [MIP-1α], and CCL8 [MCP-2]). Data were subjected to ANOVA followed by Student-Newman-Keuls (P<0.05). Laminar mRNA concentrations for all CXC chemokines were increased (P<0.05) at both the DEV and LAM horses when compared to the control horses, whereas mRNA concentrations of CCL2 and CCL8 were only increased in the LAM horses when compared to controls and the DEV horses. When taken in context with our previous studies, CXCL1, CXCL6 and CXCL8 increases precede peak laminar leukocyte accumulation. Additionally, CCL2 and CCL8 expression corroborate previous reports of monocyte/macrophage accumulation in affected laminae. Compared with previous studies, our findings demonstrate that increased laminar CXC chemokine expression consistently precedes peak leukocyte accumulation and onset of lameness in CHO laminitis models. Chemokine antagonists may be considered as possible therapeutic targets to decrease the influx of leukocytes that occurs during the development of equine laminitis.

Detection of calprotectin and apoptotic activity within the equine colon from horses with black walnut extract-induced laminitis.

The black walnut extract (BWE) model of equine laminitis is associated with a systemic inflammatory response manifest by increased expression of inflammatory cytokines in the lungs and liver as well as the laminae. The specific role of the gastrointestinal tract in development of this response is unclear and is of utmost importance, as gastrointestinal disease and laminitis are intimately related. We investigated calprotectin expression and epithelial and endothelial apoptosis in the colon of horses exposed to orally administered BWE. Sections of colon from 19 horses including 7 controls not exposed to BWE, 6 horses at the developmental time-point of leukopenia (DTP) and 6 at the onset of Obel grade 1 laminitis (LAM) after BWE-administration were histologically examined. Immunohistochemical evaluation for calprotectin expression with MAC 387 antibody was performed along with assessment of epithelial and endothelial apoptosis with caspase-3 active antibody. Calprotectin expression and percentage of apoptotic cells were compared between controls and the two treatment groups and presence of a correlation between calprotectin expression and apoptosis was evaluated. Histological findings from BWE-treated horses included eosinophil and lymphocyte epitheliotropism. The DTP group had a higher (p<0.01) calprotectin score with respect to the control group, while there was no significant difference in percentage of epithelial and endothelial apoptotic cells between groups (p=0.08 and p=0.48 respectively). No significant correlation was found between calprotectin score and epithelial or endothelial apoptosis (p=0.69 and p=0.29 respectively). There is preliminary evidence that exposure of horses to BWE results in an early inflammatory response in the colon. Further studies are needed to characterize the nature of the colonic injury in BWE-exposed horses and the link to the development of laminitis.

In vivo priming and ex vivo activation of equine neutrophils in black walnut extract-induced equine laminitis is not attenuated by systemic lidocaine administration.

Laminitis is a crippling disease of horses characterized by an inflammatory response in the tissue that suspends the axial skeleton within the hoof. Pain is a common feature of laminitic pathology and its management is an important component of the treatment regime for this disease. Systemic lidocaine administration is commonly utilized to manage pain in equine laminitis; however, the potential anti-inflammatory effects of this drug during the treatment of equine laminitis have not been investigated. Here, we sought to determine if lidocaine concentrations achieved in the plasma (therapeutic concentrations) of horses systemically administered lidocaine are capable of attenuating neutrophil activation and associated inflammation. To identify markers of activation, purified neutrophils were stimulated in vitro with LPS or recombinant equine IL-8 (reqIL-8) and surface expression of CD13 and CD18 was ascertained by immunofluorescent staining. Activation with LPS or reqIL-8 in vitro induced an elevated expression of CD13 as well as a putative conformational change in CD18 detected by elevated staining with a sub-saturating concentration of anti-CD18 mAb. Lidocaine attenuated the activation-induced changes in CD13 and CD18 expression only when used at 30-70 times therapeutic concentrations. For in vivo analyses, horses were administered black walnut extract (BWE) to induce laminitis and either systemic lidocaine (n=6) or saline (n=6) as a control. Whole blood was collected and incubated with or without reqIL-8. Following which, leukocytes were stained for CD13 and CD18. Protein was extracted from laminar tissue and subjected to gelatin zymography to measure matrix metalloproteinase-9 (MMP-9) accumulation. Results obtained show that changes in neutrophil size, granularity/complexity, CD13 surface expression and CD18 staining intensity occurred over time post BWE administration irrespective of lidocaine treatment in response to incubation alone or with 100 ng/ml of reqIL-8. The mean fluorescence intensities of neutrophils stained for either CD13 or CD18 did not differ between lidocaine treated and saline controls, nor did lamellar MMP-9 content measured by gelatin zymography. Thus, using changes in surface expression of CD13 and CD18 as markers of neutrophil activation in the horse we have shown that BWE treatment activates neutrophils in vivo and this is not affected by systemic administration of lidocaine at levels used to manage pain.

Black walnut extract: an inflammatory model.

The black walnut extract (BWE) model was developed after the discovery that horses bedded on shavings from black walnut trees commonly developed laminitis. The first investigators that consistently induced laminitis with black walnut shavings established that it was only the heartwood of the tree that induced laminitis. The BWE model of laminitis has allowed investigators to determine many of the early pathologic signaling events likely to occur in the developmental and acute clinical stages of the disease process, and has brought inflammatory injury to the forefront of laminitis research. These events must also be assessed in the carbohydrate overload models, the models that more closely reflect the clinical case of laminitis.

The pharmacologic basis for the treatment of developmental and acute laminitis.

The treatment of laminitis has been fraught with confusion and controversy for several decades, mainly because of a lack of understanding of the pathophysiology of the disease process. However, recent advances in laminitis research have greatly improved our understanding of the disease process. This article discusses the various treatment options for laminitis in the context of the findings of recent scientific investigations of laminitis pathophysiology.

Magnetic resonance imaging of the initial active stage of equine laminitis at 4.7 T.

Equine laminitis is a severely debilitating disease. There is a poor understanding of the underlying pathophysiology, and traditional imaging modalities have limited diagnostic capacity. High field strength magnetic resonance (MR) imaging allows direct visualization of the laminae, which other modalities do not. This would prove useful both in assessment of clinical patients and in further investigation into the pathophysiology of the disease. The objective of this study was to characterize the anatomic changes within the equine foot associated with the initial active stage of laminitis. Images obtained using a 4.7 T magnet were compared with digital radiographs using histologic diagnosis as the reference standard. Objective measurements and subjective evaluation for both modalities were evaluated for the ability to predict the histologic diagnosis in horses with clinical signs of laminitis as well as in clinically normal horses and horses that were in a population at risk for developing laminitis. Signal intensity and architectural changes within the corium and laminae were readily seen at 4.7 T, and there was a strong association with the histologic diagnosis of active laminitis. Measurements obtained with MR imaging were more sensitive and specific predictors of laminitis than those obtained radiographically. Subjective evaluation with MR imaging was more sensitive than with radiography and should become more specific with greater understanding of normal anatomy.

Cloning and expression of ADAM-related metalloproteases in equine laminitis.

Equine laminitis is a debilitating disease affecting the digital laminae that suspend the distal phalanx within the hoof. While the clinical progression of the disease has been well documented, the molecular events associated with its pathogenesis remain largely unknown. Using real time quantitative PCR (RT-qPCR), we have investigated the expression of genes coding for proteins containing a Disintegrin and Metalloprotease domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (tissue inhibitor of metalloprotease; TIMP) in horses with naturally-acquired (acute, chronic and aggravated chronic clinical cases) or experimentally-induced (black walnut extract (BWE) and starch gruel models) laminitis. Changes in expression of these enzymes and regulators may underlie the pathologic remodeling of lamellar tissue in laminitis. Genes encoding ADAMs involved in inflammation (ADAM-10 and ADAM-17), as well as those implicated in arthritis (ADAMTS-1, ADAMTS-4 and ADAMTS-5) were cloned, and the sequences used to generate specific oligonucleotide primers for the RT-qPCR experiments. Our results show that genes encoding ADAM-10 and ADAM-17 were not induced in most laminitic animals, whereas ADAMTS-4 gene expression was strongly upregulated in nearly all horses with experimentally-induced and naturally-acquired laminitis. The expression of matrix metalloproteases (MMP)-9 and ADAMTS-5 was also increased in many of the laminitic horses. In addition, TIMP-2 gene expression was decreased in most laminitic horses, whereas expression of genes encoding other TIMPs, namely TIMP-1 and TIMP-3, was randomly increased or decreased in the various models. We conclude that increased expression of lamellar ADAMTS-4 is a common feature of laminitis consistent with a central role of the gene product in the pathophysiology of the disease.