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.     

Type VII and XVII Collagen mRNA Expressions in Regenerated Epidermal Laminae in Chronic Equine Laminitis

To confirm ability forming the basement membrane of the regenerated laminar epidermis (rLE) in chronic laminitis, expression of type VII and type XVII collagen mRNAs in the rLE was studied applying sequences of two type of murine collagens. On northern blot analysis, complement DNA (cDNA) probes adjusted from the murine type VII and type XVII collagen could hybridize with the equine mRNAs, and each signal was detected as single-bands at approximately 9.5 kb and 5.6 kb, respectively. Contrasting with the expression level of equine glyceraldehyde-3-phosphate dehydrogenease mRNA, the band of type VII collagen mRNA in laminitis was stronger than normal, but the type XVII collagen mRNA in laminitis was less than normal. By in situ hybridization, positive signals in response to the murine type VII and type XVII collagen mRNA probes could be detected in the equine laminitic rLE region. From these results, it is concluded that the keratinocytes constructing the rLE in chronic stage of laminitis can express type VII and type XVII collagen mRNAs and these expression patterns were different from the normal.     

Decreased expression of p63, a regulator of epidermal stem cells, in the chronic laminitic equine hoof

Reasons for performing study: Abnormal epidermal stem cell regulation may contribute to the pathogenesis of equine chronic laminitis. Objective: To analyse the involvement of p63, a regulator of epidermal stem cell proliferative potential, in chronic laminitis. Methods: Epidermal tissues from skin, coronet and lamellae of the dorsal foot were harvested from 5 horses with chronic laminitis and 5 control horses. Tissues were analysed using histopathology, immunofluorescence microscopy and quantitative immunoblotting Results: Hoof lamellae of laminitic horses had a lower frequency of p63 positive cells than control lamellae, particularly in the distal region. Quantitative immunoblotting confirmed reduced p63 expression in the laminitic distal lamellar region. The decreased p63 expression in laminitic epidermal lamellae was most apparent in the abaxial region adjacent to the hoof wall and highly associated with the formation of terminally differentiated, dysplastic and hyperkeratotic epidermis in this region, whereas lamellae from control horses maintained high p63 expression throughout the axial‐abaxial axis. Conclusions: Expression of p63 in equine skin resembles that reported in other species, including man and rodents, suggesting that p63 can serve as a marker for the proliferative potential of equine epidermal stem cells. p63 expression was significantly lower in the chronic laminitic hoof than in that of control horses, suggesting laminitic hoof epithelium has more limited proliferative potential with a shift towards differentiation. This may reflect reduced activity of epidermal stem cells in laminitic hoof. It is proposed that p63 contributes to the maintenance of hoof lamellae and that misregulation of p63 expression may lead to epidermal dysplasia during lamellar wedge formation. Potential relevance: This study suggests that loss of epidermal stem cells contributes to the pathogenesis of equine laminitis. Autologous transplantation of p63‐positive epidermal stem cells from unaffected regions may have regenerative therapeutic potential for laminitic horses.     

Glucose transport in the equine hoof

Reasons for performing study: Several conditions associated with laminitis in horses are also associated with insulin resistance, which represents the failure of glucose uptake via the insulin‐responsive glucose transport proteins in certain tissues. Glucose starvation is a possible mechanism of laminitis, but glucose uptake mechanisms in the hoof are not well understood. Objectives: To determine whether glucose uptake in equine lamellae is dependent on insulin, to characterise the glucose transport mechanism in lamellae from healthy horses and ponies, and to compare this with ponies with laminitis. Methods: Study 1 investigated the effects of insulin (300 µU/ml; acute and 24 h) and various concentrations of glucose up to 24 mmol/l, on 2‐deoxy‐D‐[2,6‐³H]glucose uptake in hoof lamellar explants in vitro. Study 2 measured the mRNA expression of GLUT1 and GLUT4 transport proteins by PCR analysis in coronary band and lamellar tissue from healthy horses and ponies, ponies with insulin‐induced laminitis, and ponies suffering from chronic laminitis as a result of equine Cushing's syndrome. Results: Glucose uptake was not affected by insulin. Furthermore, the relationship between glucose concentration and glucose uptake was consistent with an insulin‐independent glucose transport system. GLUT1 mRNA expression was strong in brain, coronary band and lamellar tissue, but was weak in skeletal muscle. Expression of GLUT4 mRNA was strong in skeletal muscle, but was either absent or barely detectable in coronary band and lamellar tissue. Conclusions: The results do not support a glucose deprivation model for laminitis, in which glucose uptake in the hoof is impaired by reduced insulin sensitivity. Hoof lamellae rely on a GLUT1‐mediated glucose transport system, and it is unlikely that GLUT4 proteins play a substantial role in this tissue. Potential relevance: Laminitis associated with insulin resistance is unlikely to be due to impaired glucose uptake and subsequent glucose deprivation in lamellae.     

Chronic laminitis is associated with potential bacterial pathogens in the laminae

A common sequella of chronic laminitis in horses is repeated abscesses with variable lameness and drainage. It is unclear whether the exudate represents the debridement phase of a non-septic inflammatory process involving clearance of laminar tissue damaged during the acute episode of laminitis, or a response to a microbial infection developed by ascent of microbes from the environment to the tissue via the white line. The objective of this study was to evaluate the possibility that an undiagnosed microbial infection in laminar tissue is present in laminar tissue collected from chronically laminitic horses without an active hoof abscess. Methods to collect laminar tissue, aseptically, from control (non-laminitic) horses and those with chronic/recurrent laminitis are described. Laminae homogenates were evaluated for the presence of bacteria. Bacteria were identified using biochemical tests and sequencing of 16S rRNA and virulence genes. Laminae from chronically laminitic horses revealed 100-fold higher levels (P=0.002) of bacteria compared to control, non-laminitic horses. Although environmental organisms were identified, potential pathogens were identified. Included were Gram positive bacteria, Brevibacterium luteolum, coagulase-negative Staphylococcus spp. as well as Gram negative bacteria, enterohemorrhagic Escherichia coli and Alcaligenes faecalis. Further research is warranted to evaluate the role of bacteria in equine chronic laminitis.     

Serum markers of lamellar basement membrane degradation and lamellar histopathological changes in horses affected with laminitis

In order better to evaluate the extent to which degradation of the lamellar basement membrane (LBM) by matrix metalloproteinases (MMP) occurs in equine laminitis, we determined the concentration of type IV collagen and laminin in normal and laminitic horses, using specific immunoassays. Blood samples were obtained from both the jugular and the cephalic veins of horses (n = 10) before and after the induction of acute alimentary laminitis by carbohydrate overload. Jugular and cephalic venous blood samples were also obtained from horses affected with naturally occurring laminitis (n = 16) and nonlaminitic controls (n = 8). The serum collagen IV concentration was not changed following the induction of laminitis in the experimental group. Serum collagen IV concentration was increased in jugular venous blood obtained from cases of naturally occurring laminitis (mean +/- s.e. 218.04 +/- 18.59 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). Serum collagen IV concentration was also increased in jugular venous blood obtained from severely laminitic horses (219.50 +/- 18.18 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). A difference in serum concentration of collagen IV was not identified based on chronicity of naturally occurring laminitis. Serum laminin concentration did not differ between laminitic and nonlaminitic horses. Differences in serum laminin concentration were not identified based on sampling location (jugular or cephalic vein), severity of laminitic pain, or chronicity of spontaneous laminitis. In conclusion, the circulating concentration of collagen IV was increased in horses affected with naturally occurring laminitis. The potential role for serum collagen IV assay for characterisation of equine laminitis warrants further investigation.     

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.     

Characterisation of glucose transporter (GLUT) gene expression in broiler chickens

1. Glucose transporter (GLUT) proteins, one of which is the major insulin-responsive transporter GLUT4, play a crucial role in cellular glucose uptake and glucose homeostasis in mammals. The aim of this study was to identify the extent of mRNA expression of GLUT1, GLUT2, GLUT3 and GLUT8 in chickens intrinsically lacking GLUT4. 2. GLUT1 mRNA was detected in most tissues of 3-week-old broiler chickens, with the highest expression measured in brain and adipose tissue. GLUT2 was expressed only in the liver and kidney. GLUT3 was highly expressed in the brain. GLUT8 was expressed ubiquitously, with expression in kidney and adipose tissue relatively higher than that of other tissues. 3. Expression levels of GLUT isoforms 1, 3 and 8 in skeletal muscle tissue were very low compared to the other tissues tested. 4. [3H]Cytochalasin B binding assays on tissue from 3-week-old chickens showed that the number of cytochalasin B binding sites in skeletal muscle plasma membranes was higher than in liver plasma membranes. These results suggest that GLUT proteins and/or GLUT-like proteins that bind cytochalasin B are expressed in chicken skeletal muscles. 5. It is proposed that GLUT expression and glucose transport in chicken tissues are regulated in a manner different from that in mammals.     

In Vivo and In Vitro Evidence of the Involvement of CXCL1, a Keratinocyte-Derived Chemokine, in Equine Laminitis

Background: C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans.
Objectives: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis.
Animals: Twenty adult horses.
Methods: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leukopenia (developmental time point [DTP] group, n = 5), and at the onset of lameness (LAM group, n = 5) after BWE administration. EpCs were exposed to Toll-like/Nod receptor ligands, oxidative stress agents, and reduced atmospheric oxygen (3%). In situ PCR was used to localize the laminar cell types undergoing CXCL1 mRNA expression.
Results: Increases in laminar CXCL1 mRNA concentrations occurred in the ETP (163-fold [ P = .0001]) and DTP groups (21-fold [ P = .005]). Smaller increases in CXCL1 expression occurred in other tissues and organs. In cultured EpCs, increases ( P < .05) in CXCL1 mRNA concentration occurred after exposure to lipopolysaccharide (LPS [28-fold]), xanthine/xanthine oxidase (3.5-fold), and H₂O₂ (2-fold). Hypoxia enhanced the LPS-induced increase in CXCL1 mRNA ( P = .007). CXCL1 gene expression was localized to laminar EpCs, endothelial cells, and emigrating leukocytes.
Conclusion and Clinical Importance: These findings indicate that CXCL1 plays an early and possibly initiating role in neutrophil accumulation in the BWE laminitis model, and that laminar keratinocytes are an important source of this chemokine. New therapies using chemokine receptor antagonists may be indicated.     

Characterisation and distribution of epidermal growth factor receptors in equine hoof wall laminar tissue: comparison of normal horses and horses affected with chronic laminitis

Epidermal growth factor (EGF) receptors were detected in plasma membrane preparations of equine hoof wall laminar tissue at concentrations comparable to that of equine liver. Scatchard analysis of the equilibrium binding data suggested the presence of two classes of EGF binding sites in most of the controls (plasma membranes from clinically normal horses); a high-affinity class and a more numerous low-affinity class. The dissociation constant of the low-affinity class of EGF-specific receptors (KD = 1 x 10(-9)M) is in reasonable agreement with other values established for the EGF receptor. The variability between individual estimates for the KD of the high-affinity receptor class precluded an accurate estimate for those sites. A possible explanation is discussed. The high-affinity binding sites were uniformly absent in plasma membranes prepared from horses affected by chronic laminitis. Autoradiographic analysis localised the EGF receptors primarily to the secondary epidermal laminae, with an apparent greater density over the proliferative basal keratinocytes. Little label was associated with the dermal or the keratinised primary epidermal laminae. Tissue from horses with chronic laminitis had EGF receptors located uniformly over the hyperplastic epidermal keratinocytes. These data suggest that an EGF-mediated response may be involved in the hyperproliferative response that is characteristic of chronic laminitis.     

Endothelin mediated contraction of equine laminar veins

REASONS FOR PERFORMING STUDY: Endothelin-1 (ET-1) may be a key mediator in the pathogenesis of laminitis, but endothelin-mediated responses in the venous microcirculation of the equine foot have yet to be fully characterised. OBJECTIVES: To characterise the response of equine laminar veins to ET-1 and evaluate the ET-1 receptor subtypes that mediate this response. METHODS: Small veins (150-500 microns) draining the equine digital laminae from healthy horses and ponies subjected to euthanasia at an abattoir were investigated using wire myography. Concentration response curves were constructed for ET-1 in the presence of ETA (BQ123) and ETB (BQ788) receptor antagonists, and L-NAME, a nitric oxide synthase blocker. The selective ETB receptor agonist BQ3020 was investigated alone and following incubation with L-NAME, with or without BQ788. RESULTS: Endothelin-1 contraction of laminar veins was significantly inhibited by BQ123 but not by BQ788. In the presence of L-NAME, sensitivity of laminar veins to ET-1 was enhanced 4-fold, and further addition of BQ788 did not alter this increased sensitivity. BQ3020 induced no venoconstriction; however, in the presence of L-NAME, it caused contraction of veins with approximately 30% of the efficacy of ET-1. The action of BQ3020 in the presence of L-NAME was abolished by BQ788. CONCLUSIONS: Both ETA and ETB receptors are involved in the net tonic response to ET-1 in normal laminar veins. A population of ETB receptors may be present on the vascular endothelium and on smooth muscle of laminar veins, and the action of ET-1 at these 2 sites is likely to be approximately equal and opposite. POTENTIAL RELEVANCE: Our results clarify the function of the ET-1 receptor subtypes in laminar veins from healthy horses. Further study of ET-1 receptors in laminitic horses is therefore warranted.     

Unilateral basement membrane zone alteration of the regenerated laminar region in equine chronic laminitis

Between the laminar epidermis and the laminar dermis of laminar region (LR) in equine foot, it can be observed the basement membrane zone (BMZ), which is composed of a basement membrane and its accompaniments like the hemidesmosome and anchoring fibril. Alteration in the BMZ in equine laminitis is possibly related with not only development but also recovery outcome and recurrence of this disease. However, there is little known about the structure of the BMZ during the recovery phase of this disease. To assess the condition of the BMZ of LR affected by chronic laminitis, the tissue was examined in three cases at two weeks, four weeks and three months after the onset of laminitis, using pathological, immunohistochemical and electron microscopic techniques. Histologically in all laminitis cases, there was a regenerated laminar epidermis with proliferating keratinocytes between the Stratum medium and the dermis, but it included the undeveloped secondary epidermal laminae (ud-SELs) structure in one side of the primary epidermal laminae, especially in the part of the deep area of LR. Immunohistochemical results were positive for the anti-type IV collagen, anti-type VII collagen and anti-laminin 5 antibodies in the most BMZs. However, partial BMZs adjacent to the ud-SELs were negative for the anti-type VII collagen and anti-laminin 5 antibodies. Ultrastructurally, in the BMZ of the ud-SEL, the lamina densa and the lamina lucida were present. In contrast, the anchoring fibrils and the hemidesmosomes were either absent, or present at lower than normal levels. In conclusion, the present study indicated that the part of regenerated LR in chronic laminitis was not able to fully restore to construct the BMZ for a long time, especially in the unilateral side of laminar epidermis. It might be related with recurrence of this disease.     

Rehabilitating the Chronically Laminitic Foot

Trimming and shoeing the equine patients suffering with chronic laminitis entails a well organized approach, improves quality of life and generally results in a better outcome. The chronic laminitic hoof presents clinically in a variety of ways and thus treatment needs to be tailored to the individual horse.     

Evidence for vascular and enzymatic events in the pathophysiology of acute laminitis: which pathway is responsible for initiation of this process in horses?

To date, there is a substantial amount of data to support the hypotheses that vascular and enzymatic changes are ongoing in experimental laminitis. Furthermore, there is substantial in vitro evidence that the enzymatic changes weaken the dermo-epidermal attachments leading to mechanical failure of the hoof-bone interface of the equine digit. However, investigators of both the vascular and enzymatic theories have, to date, been unable to substantiate the effects of these pathophysiological changes in vivo on laminar tissues of horses afflicted with experimentally induced or naturally acquired laminitis. In addition, the effects of laminitis-inducing treatment have not been prevented or reversed by treatment with an MMP inhibitor or a vasoactive antagonist. It is possible that there is simultaneous activation of the vascular and enzymatic pathways and/or other inflammatory processes. Moreover, the third theory involving mechanical factors cannot be discounted simply because strong evidence for vascular and enzymatic changes exists. It is common for horses with severe musculoskeletal disease affecting weightbearing on a limb to develop laminitis in the contralateral limb. It remains to be determined what factors are responsible for initiation of laminitis in these individuals. Evidence has not been presented that precludes the possibility of coincident occurrence of vascular and enzymatic changes. In fact, many of the inflammatory mediators (e.g. interleukin-1beta) found in laminitic tissues can concurrently stimulate synthesis of vasoactive substances and activate MMPs. Because enzymatic action on proteins is largely dependent on the concentrations of proteins and enzyme, the enzymatic theory is not dependent upon increased delivery of enzymes via increased capillary flow. Likewise, because vascular changes can alter tissue function via increased capillary flow and oedema formation, the vascular theory is not dependent upon decreased capillary flow. It is true that naturally acquired laminitis is widely variable in severity and predisposing diseases. Therefore, most probably there are multiple mechanisms involved in the initiation and propagation of the pathophysiologic cascade(s) and, therefore, successful intervention will necessitate multiple treatment modalities.     

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.     

Proinflammatory cytokine responses of cultured equine keratinocytes to bacterial pathogen‐associated molecular pattern motifs

Reasons for performing study: Further knowledge of equine keratinocyte physiology and keratinocyte response to various stimuli is important in developing a better understanding of disease states involving the epidermis. Objectives: To assess the inflammatory cytokine response of cultured equine keratinocytes to various pathogen‐associated molecular pattern molecules (PAMPs) from both Gram‐negative and positive bacteria likely to be present in equine sepsis. Methods: Keratinocytes were isolated from skin of 2 horses and primary cultures performed. Keratinocytes were harvested for RNA extraction after exposure to lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), bacterial DNA (CpG), flagellin or maintained in medium (controls) for 4 or 24 h. Real time‐quantitative PCR was used to quantify interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and CXCL8 mRNA concentrations. Results: Increases (P<0.05) in IL‐1β, IL‐6 and CXCL8 mRNA concentrations were induced by LPS exposure compared to controls. Increased mRNA concentrations of both IL‐6 and CXCL8 were also noted (vs. controls) upon exposure to flagellin. Overall, responses were greater at 4 h. No increases (P>0.05) in cytokine expression by keratinocytes were present after LTA, PGN or CpG exposure. Conclusions: Increased proinflammatory cytokine expression in response to LPS and flagellin indicate that equine keratinocytes have functional TLR4 and TLR5 receptor signalling. However, the lack of keratinocyte stimulation by PGN, LTA or CpG provides no evidence for functional TLR2, TLR9 or NOD receptor signalling. These results suggest that equine keratinocytes are more responsive to PAMPs usually associated with Gram‐negative sepsis and unresponsive to PAMPs most commonly associated with Gram‐positive sepsis. Potential relevance: The increased incidence of injury of epidermal structures in clinical cases of Gram‐negative (vs. Gram‐positive) sepsis in the horse may be due to a lack of functional TLR signalling for Gram‐positive PAMPs in the equine keratinocyte.     

Black walnut extract-induced laminitis in horses is associated with heterogeneous dysfunction of the laminar microvasculature

REASONS FOR PERFORMING STUDY: Equine laminitis purportedly involves haemodynamic dysfunction at the level of the laminar vasculature. However, to date, no studies have been performed characterising the function of laminar arteries and veins during the prodromal stages of equine laminitis. HYPOTHESIS: That the prodromal stages of laminitis are associated with contractile dysfunction of the equine laminar vasculature. OBJECTIVE: To assess contractile function of laminar arteries and veins to phenylephrine (PE) and 5-hydroxytryptamine (5-HT). METHODS: Horses were administered black walnut heartwood extract (BWHE) or water (control horses) via nasogastric intubation. After euthanasia, laminar vessels (100-800 microm internal diameter) were isolated and mounted on small vessel myographs to assess contractile function. RESULTS: Contractile responses to PE or 5-HT were identical in laminar arteries isolated from either control horses or those administered BWHE. In contrast, responses to PE or 5-HT were significantly reduced in laminar veins isolated from BWHE-administered horses when compared with laminar veins isolated from control horses. CONCLUSIONS AND POTENTIAL RELEVANCE: These results are consistent with the prodromal stages of laminitis being associated with selective dysfunction of laminar veins. Further studies are required to discern the precise nature of this dysfunction and its potential relevance to the pathogenesis of acute laminitis in the horse and possible therapeutic targets for treatment.     

Expression of interleukin-1beta in the digital laminae of horses in the prodromal stage of experimentally induced laminitis

OBJECTIVE: To study expression of interleukin-1beta (IL-1beta) in the digital laminae of horses in the prodromal stage of experimentally induced laminitis. ANIMALS: 8 healthy adult horses with no signs of laminitis. PROCEDURES: Black walnut extract was administered via nasogastric tube to 4 horses, and water was administered to the remaining 4 (controls). Complete blood counts and physical examinations were performed every 30 minutes after administration of black walnut extract or water. General anesthesia was induced when total WBC count decreased by 30% in horses given the black walnut extract and 3 hours after water administration in control horses. The left forefoot was perfusion fixed with neutral-buffered 10% formalin, and paraffin-embedded sections of the digit were used for in situ hybridization with an equine-specific IL-1beta probe. RESULTS: IL-1beta mRNA expression was observed in perivascular cells of the small laminar venules and capillaries in all 4 horses given black walnut extract and in interstitial cells remote from the microvasculature in 1 of the 4. Other cellular components of the laminar tissue and cellular components of the digital arterioles and veins did not exhibit IL-1beta mRNA expression. Expression of IL-1beta mRNA was not detected in laminae from control horses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that IL-1beta mRNA is expressed by perivascular cells in the laminar tissues of horses in the prodromal stage of experimentally induced laminitis. This provides evidence of an inflammatory process during the prodromal stage of laminitis, indicating that local digital proinflammatory cytokine expression may be an initiating factor in laminitis.     

Differential regulation of the GLUT1 and GLUT3 glucose transporters by growth factors and pro-inflammatory cytokines in equine articular chondrocytes

Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-d-[2,6-3H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-alpha (100 ng mL(-1)), IL-1beta (100 ng mL(-1)), IGF-I (20 ng mL(-1)), TGF-beta (20 ng mL(-1)) and insulin (12.5 microg mL(-1)) before measuring uptake of non-metabolizable 2-deoxyglucose in the presence and absence of the glucose transport inhibitor cytochalasin B. Polyclonal antibodies to GLUT1 and GLUT were used to compare GLUT1 and GLUT3 expression in stimulated and un-stimulated alginate encapsulated chondrocytes by Western blotting. Results indicated that 2-deoxyglucose uptake was inhibited by up to 95% in the presence of cytochalasin B suggesting that glucose uptake into equine chondrocytes is GLUT-mediated. Insulin had no effect on glucose uptake, but treatment with IGF-I, TGF-beta, IL-1beta and TNF-alpha resulted in a significant increase (>65%) in 2-deoxyglucose uptake compared to control values. GLUT1 was found to be increased in chondrocytes stimulated with all the growth factors and cytokines but GLUT 3 was only upregulated by IGF-I. The data presented support a critical role for glucose in the responses of equine articular chondrocytes to pro-inflammatory cytokines and anabolic endocrine factors.