Laminar inflammatory gene expression in the carbohydrate overload model of equine laminitis

Reasons for performing study: There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis‐related laminitis than the black walnut extract (BWE) model. Objectives: To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. Methods: Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature >38.9°C (DEV group, n = 8) or at onset of Obel grade 1 lameness (OG1 group, n = 8). Control horses (CON group, n = 8) were anaesthetised 24 h after NG administration of 6 l of deionised water. Laminar tissue was collected from horses while under anaesthesia, followed by humane euthanasia. Real time‐quantitative PCR was used to assess laminar mRNA concentrations of genes involved in inflammatory signalling. Results: Increased mRNA concentrations (P<0.05) for IL‐1β, IL‐6, IL‐12p35, COX‐2, E‐selectin and ICAM‐1 were present in laminae from horses with OG1 lameness but not at the DEV time, when compared to the CON horses. No differences between the groups were found for IL‐2, IL‐4, IL‐10, TNF‐α, IFN‐γ or COX‐1 at either the DEV or OG1 time points. Conclusions: There was a notable difference in the temporal pattern of inflammatory events between the BWE and CHO models, with the majority of laminar inflammatory events appearing to occur at or near the onset of lameness in the CHO model, whereas many of these events peak earlier in the developmental stages in the BWE model. This suggests that, in addition to circulating inflammatory molecules, there may be a local phenomenon in the CHO model resulting in the simultaneous onset of multiple laminar events including endothelial activation, leucocyte emigration and proinflammatory cytokine expression. Potential relevance: The similar (although somewhat delayed) inflammatory response in the CHO model of laminitis indicates that inflammatory signalling is a consistent entity in the pathophysiology of laminitis.     

Intestinal bacterial overgrowth includes potential pathogens in the carbohydrate overload models of equine acute laminitis

Carbohydrate overload models of equine acute laminitis are used to study the development of lameness. It is hypothesized that a diet-induced shift in cecal bacterial communities contributes to the development of the pro-inflammatory state that progresses to laminar failure. It is proposed that vasoactive amines, protease activators and endotoxin, all bacterial derived bioactive metabolites, play a role in disease development. Questions regarding the oral bioavailability of many of the bacterial derived bioactive metabolites remain. This study evaluates the possibility that a carbohydrate-induced overgrowth of potentially pathogenic cecal bacteria occurs and that bacterial translocation contributes toward the development of the pro-inflammatory state. Two groups of mixed-breed horses were used, those with laminitis induced by cornstarch (n=6) or oligofructan (n=6) and non-laminitic controls (n=8). Cecal fluid and tissue homogenates of extra-intestinal sites including the laminae were used to enumerate Gram-negative and -positive bacteria. Horses that developed Obel grade2 lameness, revealed a significant overgrowth of potentially pathogenic Gram-positive and Gram-negative intestinal bacteria within the cecal fluid. Although colonization of extra-intestinal sites with potentially pathogenic bacteria was not detected, results of this study indicate that cecal/colonic lymphadenopathy and eosinophilia develop in horses progressing to lameness. It is hypothesized that the pro-inflammatory state in carbohydrate overload models of equine acute laminitis is driven by an immune response to the rapid overgrowth of Gram-positive and Gram-negative cecal bacterial communities in the gut. Further equine research is indicated to study the immunological response, involving the lymphatic system that develops in the model.     

Production of amines in equine cecal contents in an in vitro model of carbohydrate overload

Acute laminitis can be induced experimentally in horses by the administration of carbohydrate, resulting in fermentation within the cecum and ischemia-reperfusion of the digits. The products of fermentation that trigger acute laminitis are as yet unknown; however, compounds such as amines might play a role due to their potential vasoactive properties. The objectives of this study were to quantify the amines present in equine cecal contents and to use a model of carbohydrate overload in vitro to test the hypothesis that carbohydrate fermentation is associated with increased amine production. Cecal contents from each horse were divided into aliquots and incubated anaerobically with either cornstarch or inulin (a form of fructan carbohydrate; both 1 g/100 mL). The pH was measured and samples were taken at the same time for amine measurement by HPLC at 2-h intervals over a 24-h period. In a second set of experiments, the effects of the antibiotic virginiamycin (1 mg/100 mL), calcium (CaPO4; 0.3 g/100 mL), and plant steroidal saponin (Yucca schidigera extract; 0.1 g/100 mL) were examined on pH and amine concentrations in cecal contents incubated with starch or inulin. Both starch and inulin caused significant time-dependent falls in pH, from 6.7 +/- 0.1 at 0 h to 5.2 +/- 0.1 (starch) and 5.0 +/- 0.1 (inulin) at 24 h. Fermentation of carbohydrate was also associated with increased production of phenylethylamine and isoamylamine (two- to threefold increases) as well as putrescine and cadaverine (1.5- to twofold increases). Virginiamycin inhibited the fall in pH and increases in production of phenylethylamine and isoamylamine, while calcium phosphate moderated the changes in pH only. Yucca schidigera extract was without effect. These data show that fermentation of carbohydrate by equine cecal microbiota may lead to increased production of amines.     

Reference gene screening for analyzing gene expression in the heart,liver, spleen,lung and kidney of forest musk deer

The screening of reference genes for real-time quantitative PCR (qPCR) in forest musk deer (FMD) tissue is of great significance to the basic research on FMD. However, there are few reports on the stability analysis of FMD reference genes so far. In this study, We used qPCR to detect the expression levels of 11 reference gene candidates (18S rRNA, beta-actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box-binding protein [TBP], hypoxanthine phosphoribosyltransferase 1 [HPRT1], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide [YWHAZ], hydroxymethylbilane synthase [HMBS], eukaryotic translation elongation factor 1 alpha 1 [EEF1A1], succinate dehydrogenase complex flavoprotein subunit A [SDHA], peptidylprolyl isomerase B [PPIB], and ubiquitin C [UBC]) in heart, liver, spleen, lung and kidney of FMD. After removing 18S rRNA on account of its high expression level, geNorm, NormFinder, BestKeeper and ΔCt algorithms were used to evaluate the expression stability of the remaining genes in the five organs, and further comprehensive ranking was calculated by RefFinder. According to the results, the selected reference genes with the most stable expression in the heart of FMD are SDHA and YWHAZ, while in the liver are ACTB and SDHA; in the spleen and lung are YWHAZ and HPRT1; in the kidney are YWHAZ and PPIB. The use of common reference genes in all five organs is not recommended. The analyses showed that tissue is an important variability factor in genes expression stability. Meanwhile, the result can be used as a reference for the selection of reference genes for qPCR in further study.     

Effects of endotoxaemia and carbohydrate overload on glucose and insulin dynamics and the development of laminitis in horses

Reasons for performing study: Insulin resistance (IR) is a risk factor for pasture‐associated laminitis in equids and alimentary carbohydrate overload may trigger laminitis. Whether glucose metabolism responses to carbohydrate overload are more pronounced in insulin‐resistant horses requires further study. Hypothesis: Horses pretreated with endotoxin to alter insulin sensitivity differ significantly in their glucose and insulin responses to carbohydrate overload. Methods: Horses (n = 24) were divided into 3 groups. A lipopolysaccharide (LPS; n = 8) group that received endotoxin as an 8 h 7.5 ng/kg bwt/h i.v. continuous rate infusion, an oligofructose (OF; n = 8) group that received an infusion of saline followed by 5 g/kg bwt OF via nasogastric intubation, and a LPS/OF (n = 8) group that received LPS followed 16 h later by OF. Glucose and insulin dynamics were evaluated at ‐24 h and 48 h using the frequently sampled i.v. glucose tolerance test and minimal model analysis. Physical examinations and haematology were performed and the severity of laminitis assessed. Results: Horses receiving LPS developed leucopenia and both LPS and OF induced clinical signs consistent with systemic inflammation. Insulin sensitivity significantly decreased (P<0.001) over time, but responses did not differ significantly among groups. Time (P<0.001) and treatment × time (P = 0.038) effects were detected for the acute insulin response to glucose, with mean values significantly increasing in LPS and LPS/OF groups, but not the OF group. Five horses in the LPS/OF group developed clinical laminitis compared with 0 and 2 horses in the LPS and OF groups, respectively. Conclusions: Endotoxaemia and carbohydrate overload reduce insulin sensitivity in horses. Endotoxin pretreatment does not affect the alterations in glucose metabolism induced by carbohydrate overload. Potential relevance: Insulin sensitivity decreases after carbohydrate overload in horses, which may be relevant to the development of pasture‐associated laminitis.     

Cyclooxygenase expression in the early stages of equine laminitis: a cytologic study

BACKGROUND: Recent reports indicate increased amounts of mRNA from inflammation-related genes in the prodromal stage of laminitis. HYPOTHESIS: Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) undergo distinct patterns of expression in equine laminae in the developmental stage (DEV) and acute clinical stage (LAM) of laminitis. ANIMALS: Horses selected from an outbred population were placed into 1 of 4 groups: DEV (n = 5), CON-3h (control group for DEV, n = 5), LAM (n = 5) and CON-10h (control group for LAM, n = 5). METHODS: Laminar and skin samples were obtained from (1) animals either undergoing leukopenia (DEV) or the onset of clinical signs of laminitis (LAM) after black walnut extract (BWE) administration and (2) animals either 3 (CON-3h) or 10 (CON-10h) hours after administration of water. Real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunohistochemical analysis were performed for COX-1 and COX-2. RESULTS: Upon immunohistochemical analysis of all 4 groups, COX-2 was expressed by most viable epithelial cells in both laminae and skin. COX-1 exhibited similar epithelial expression to COX-2 in skin epidermis, but was expressed exclusively in the basal layer of laminar epidermis. COX-1 protein was not detectable in dermal vasculature of equine skin or laminae, whereas COX-2 was present in endothelial and vascular smooth muscle cells of dermal vasculature in both skin and laminae in all groups. A marked increase in laminar COX-2 protein concentrations was detected on immunoblotting in the DEV group, although a lesser increase was observed in the LAM group. CONCLUSIONS AND CLINICAL IMPORTANCE: COX-2 protein expression is markedly increased in the resident laminar cell types in the developmental stage of BWE-induced laminitis.     

Dietary fructan carbohydrate increases amine production in the equine large intestine: implications for pasture-associated laminitis

Pasture-induced laminitis in the horse is associated with the overconsumption of fermentable carbohydrate, in the form of simple sugars, fructans, or starch. The fermentation of carbohydrate in the cecum and large intestine results in the production of lactic acid and other toxins or "laminitis trigger factors." Vasoactive amines have been suggested as possible initiating factors. The aim of this study was to feed a commercially available form of fructan carbohydrate (inulin, 3 g/kg of BW per day) to normal ponies and to ponies predisposed to laminitis, to mimic a change from a basal hay diet to lush spring-summer pasture. Five normal and 6 laminitis-prone, native-breed ponies were acclimated to a basal hay diet before the inclusion of inulin and chopped dried grass. Blood samples, fecal samples, and foot temperature measurements were taken throughout the study. Amines were measured in the feces and plasma by HPLC and liquid chromatography-mass spectrometry, respectively. The pH of the fecal samples decreased from 6.89 +/- 0.11 on the hay diet to a minimum of 6.18 +/- 0.11 with the addition of inulin (P <0.05). An increase was observed in the fecal concentrations of a number of amines, including tryptamine (2.5-fold increase, P <0.05) and tyramine (2-fold increase, P <0.05). No changes were noted in plasma amine concentrations or plasma D- or L-lactate, indicating that there may be a threshold of hindgut pH change before mucosal damage can result in the release of these factors into the circulation. No differences in pH or any of the measured compounds were observed between the group of normal ponies and those predisposed to laminitis. This indicates that differences in the intestinal microflora do not account for this predisposition. However, the results from this study indicate that moderate increases in dietary fructan carbohydrate can produce increases in bacterial fermentation products and other compounds in the large intestine, which may be relevant to the pathogenesis of acute laminitis in ponies on pasture.     

Glucocorticoid therapy and the risk of equine laminitis

Although glucocorticoids have been used successfully for the treatment of noninfectious inflammatory diseases of horses for more than 35 years, their use has been attended by a fear of the induction of laminitis. This paper reviews the evidence for this fear and the possible mechanisms whereby glucocorticoids could participate in laminitis induction. Although the association of laminitis with elevated serum cortisol in pituitary pars intermedia dysfunction suggests that chronic exposure to glucocorticoids may be part of laminitis pathogenesis, review of published reports and databases suggests that glucocorticoid‐induced laminitis is a relatively rare occurrence. However, several of the actions of glucocorticoids are similar to those known to be involved in laminitis pathogenesis. Glucocorticoid administration can induce insulin resistance, lead to vascular dysfunction that potentiates vasoconstriction, and interfere with keratinocyte proliferation and differentiation as well as matrix integrity, all mechanisms that could possibly induce laminitis. Drug formulation, dose and route of administration, and the systemic and hoof disease history of the horse must all be considered when assessing laminitis risk during glucocorticoid treatment. Generally, local glucocorticoid administration presents little risk as does systemic treatment of recurrent airway obstruction without concurrent disease. Caution should be used however in horses that are overweight and/or insulin resistant, or have had a recent bout of acute laminitis of alimentary or endotoxic origin. Overall, however, the risk of laminitis after glucocorticoid treatment, especially local use, is acceptable compared to the many benefits of these drugs.     

Bovine laminitis: clinical aspects, pathology and pathogenesis with reference to acute equine laminitis

This review deals with the features of clinical and subclinical laminitis in cattle. Prominent clinical signs of acute laminitis are a tender gait and arched back. The sole horn reveals red and yellowish discolourations within five days. In subacute and chronic cases clinical signs are less severe. In chronic laminitis the shape of the claws is altered. Laminitis is frequently followed by sole ulceration and white zone lesions. Blood tests showed no significant changes for laminitic animals. Arteriographic studies of claws affected by laminitis indicated that blood vessels had narrowed lumens. Gross pathology revealed congestion of the corium and rotation of the distal phalanx. Histopathologic studies indicate that laminitis is associated with changes of the vasculature. Peripartum management and nutrition are important factors in its aetiology. It is hypothesised that laminitis is evoked by disturbed digital circulation. In the pathogenesis of acute laminitis three factors are considered important: the occurrence of thrombosis, haemodynamic aspects of the corium, and endotoxins which trigger these pathologic events.     

Expression of endothelin in equine laminitis.

Biosynthesis of endothelin-1 (ET-1), the most potent endogenous vasoconstrictor yet identified, is increased following myocardial infarction (MI) in man. Pathological events which occur in the connective tissues of the equine hoof during laminitis are similar in some respects, to changes occurring in the myocardial connective tissues following MI in man. The objective of this study was to determine whether ET-1 expression in connective tissues obtained from the hoof of laminitic horses is increased compared with tissues obtained from healthy horses. Expression of ET-1 in connective tissues of the equine hoof was measured following tissue extraction from 3 groups of horses: horses in which acute laminitis had been induced by the administration of starch; chronically foundered horses; nonlaminitic horses. The concentration of ET-1 in laminar connective tissues obtained from all laminitic horses (1573.0 +/- 392.8 pg/g of tissue; n = 10) was increased when compared with tissues obtained from nonlaminitic horses (392.5 +/- 117.4 pg/g of tissue; n = 5) (P<0.05). The concentration of ET-1 in laminar connective tissues obtained from the experimentally induced, acute laminitic horses (1043.6 +/- 254.4 pg/g of tissue; n = 7) and from the spontaneously affected, chronic laminitic horses (2808.3 +/- 878.6 pg/g of tissue; n = 3) was increased compared with the control group (P<0.05, P<0.01, respectively). The concentration of ET-1 in laminar connective tissues obtained from the chronic laminitic horses was greater than that of the experimentally induced, acute laminitic group (P<0.05). It is suggested that the data provide a strong argument that increased ET-1 expression in the connective tissues of the equine hoof represent a potentially important and hitherto unrecognised component of the pathophysiology of equine laminitis. Further studies are needed to determine whether inhibitors of ET-1 converting enzyme or antagonists of ET-1 receptors might be useful in the treatment and prevention of laminitis in horses.     

Copper, zinc and manganese concentrations in equine liver, kidney and plasma

Five groups of horses were fed different diets of known trace mineral concentration for a minimum of six months. Copper (Cu), zinc (Zn) and manganese (Mn) concentrations were measured in livers of 125 yearling horses and kidneys of 81 yearling horses as an assessment of trace mineral status. Plasma Cu and Zn determinations were made for all horses.

Mean hepatic Cu concentrations of horses fed diets containing 6.9 to 15.2 mg Cu/kg dry matter (DM) feed were 0.27 to 0.33 μmol/g DM tissue. Plasma Cu concentrations ranged between 22.8 to 28.3 μmol/L. There was no simple mathematical relationship between plasma and hepatic Cu concentrations. Mean hepatic Zn concentrations in horses fed diets containing 25.6 to 52.2 mg Zn/kg DM feed were determined to be between 2.75 to 2.91 μmol/g DM tissue. Mean plasma Zn concentrations in groups of horses were between 11.7 to 13.5 μmol/L. Plasma Zn concentrations were not indicative of hepatic Zn concentration. Hepatic Mn concentrations ranged between 0.13 and 0.14 μmol/g DM tissue.

Renal Zn concentrations ranged between 1.55 to 1.63 μmol/g DM tissue and did not differ with diet. Mean renal Mn concentrations were 0.09 μmol/g DM tissue for all groups of horses. Renal Cu concentrations ranged from 0.36 to 0.47 μmol/g DM tissue and differed with diet.

     

Tissue-specific dysregulation of cortisol metabolism in equine laminitis

REASONS FOR PERFORMING STUDY: The role of glucocorticoids (GCs) in the pathogenesis of laminitis is incompletely understood. Local tissue activity of GC is regulated by the steroid converting enzyme, 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1). Changes in integumentary (skin and hoof lamellar) 11beta-HSD activity occurring during laminitis could affect the extent to which GCs are involved in its development. HYPOTHESIS: That changes in integumentary 11beta-HSD-1 activity associated with the laminitic condition would lead to elevated local tissue levels of GCs, which could subsequently contribute, through paracrine and autocrine mechanisms, to the further development of laminitis; and that similar changes in 11beta-HSD-1 activity would be evident in both skin and hoof lamellar tissue. METHODS: Activity of 11beta-HSD-1 was determined in skin and hoof lamellar tissue specimens obtained from normal and laminitic horses using a radiometric assay. Skin samples were obtained from 10 normal horses and from 10 horses before and after induction of acute laminitis following administration of starch via nasogastric tube. Hoof lamellar samples were obtained from 10 normal horses, 10 horses following induction of acute laminitis and 4 chronically-foundered horses. Bidirectional 11beta-HSD-1 activity was measured in both skin and lamellar tissues. RESULTS: 11-ketoreductase activity exceeded 11beta-dehydrogenase activity in both skin and lamellar tissues. Cutaneous activity was higher than lamellar 11beta-HSD-1 activity in all groups. Both ketoreductase and dehydrogenase activity increased in skin and lamellae following experimental induction of acute laminitis, but the increase in ketoreductase activity was substantially greater than that for dehydrogenase in the lamellae. Induction of acute laminitis was attended by increases of 227 and 220% in cutaneous dehydrogenase and ketoreductase activity, respectively, and 173 and 398% in lamellar dehydrogenase and ketoreductase activity, respectively (P<0.05). CONCLUSIONS: The 11-ketoreductase moiety of 11beta-HSD-1 plays a role in equine skin and hoof lamellae regarding the regulation of local glucocorticoid activity. Increased 11-ketoreductase activity will lead to increased local tissue GC activity by virtue of conversion of cortisone to cortisol. POTENTIAL RELEVANCE: The laminitic condition is attended by integumentary biochemical changes that enhance the local concentration of cortisol, especially in the hoof lamellar interface. Through multiple and diverse actions, increased local GC activity contributes to the pathogenesis and morbidity associated with laminitis. Pharmacological manipulation of 11beta-HSD-1 deserves further investigation regarding the prevention and treatment of laminitis.     

Differential gene expression of CYP3A isoforms in equine liver and intestines

Tydén, E., Löfgren, M., Pegolo, S., Capolongo, F., Tjälve, H., Larsson, P. Differential gene expression of CYP3A isoforms in equine liver and intestines. J. vet. Pharmacol. Therap.  35 , 588–595. Recently, seven CYP3A isoforms – CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP129 – have been isolated from the horse genome. In this study, we have examined the hepatic and intestinal gene expression of these CYP3A isoforms using TaqMan probes. We have also studied the enzyme activity using luciferin‐isopropyl acetal (LIPA) as a substrate. The results show a differential gene expression of the CYP3A isoforms in the liver and intestines in horses. In the liver, CYP3A89, CYP3A94, CYP3A96 and CYP3A97 were highly expressed, while in the intestine there were only two dominating isoforms, CYP3A93 and CYP3A96. The isoform CYP3A129 was not detected in the liver or the intestine, although this gene consists of a complete set of exons and should therefore code for a functional protein. It is possible that this gene is expressed in tissues other than the liver and intestines. In the intestine, both CYP3A96 and CYP3A93 showed the highest gene expression in the duodenum and the proximal parts of the jejunum. This correlated with a high protein expression in these tissues. Studies of the enzyme activity showed the same K_m for the LIPA substrate in the liver and the intestine, while the maximum velocity (V_max) in the liver was higher than in the intestine. Our finding of a differential gene expression of the CYP3A isoforms in the liver and the intestines contributes to a better understanding of drug metabolism in horses.     

Insulin resistance in equine digital vessel rings: an in vitro model to study vascular dysfunction in equine laminitis

Reasons for performing study: One of the causes of equine laminitis is hyperinsulinaemia, which may be associated with endothelial dysfunction and insulin resistance of vessels. Hypothesis and objectives: Insulin resistance can be induced in palmar digital vessels by continued exposure to insulin in vitro. The objective was to evaluate this in vitro model for future studies. Methods: Palmar digital vessel segments were collected immediately after euthanasia from horses with normal insulin/glucose blood values. Four arterial and 4 venous rings (3 mm wide) were prepared and each ring mounted in a tissue bath, containing Tyrode's solution at 37°C, 2 g tension was applied and the rings allowed to equilibrate for 45 min. Of the 4 rings of each vessel type, one was used as a control. One each of the remaining 3 rings was used for incubation with insulin (to induce resistance), wortmannin (to block PI3‐kinase) and PD‐098059 (to block MAP‐kinase), respectively, for 30 min. After the incubation period, the rings were contracted with phenylephrine. When the response reached a plateau, a single dose of insulin was added to the baths and the response of each ring monitored for 30 min. Results: Insulin relaxed the control rings and those treated with PD 098059 but contracted those pretreated with insulin and wortmannin. Normal relaxation responses of the rings were converted to contractions by insulin resistance. Insulin resistance was confirmed by the qualitative response of insulin‐incubated and wortmannin‐incubated rings. Conclusions: This study demonstrated successful induction of insulin resistance in both arterial and venous rings. It also suggested that the MAP‐kinase pathway plays a minor role in controlling vasomotor tone under normal physiological conditions. Potential relevance: The study suggests that the induction of insulin resistance in equine palmar digital vessel rings is reliable and provides a good in vitro model for studying the vascular insulin resistance which may occur in equine laminitis.