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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‐1β, 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‐1β, 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.

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

6-week-old, Arabian-cross male foal was referred to the Veterinary Teaching Hospital at the Louisiana State University School of Veterinary Medicine for a 1-week history of progressive weakness and intermittent right thoracic limb lameness. The foal had a normal birth, was healthy, had been active, and had not exhibited any abnormalities of gait before presentation. The current owner had acquired the mare just before foaling. The foal presented in sternal recumbency and was unable to rise without assistance. Physical examination identified a quiet, moderately responsive foal with dull mentation and body weight of 64 kg. Rectal temperature was 101.0°F, heart rate was 64 beats/min, and respiratory rate was 28 breaths/min. Mucous membranes were tacky, there was mild decreased skin turgor, and capillary refill time (CRT) was 2 seconds. There was a grade II/VI holosystolic murmur over the left heart base that was considered functional. Increased bronchovesicular sounds were present bilaterally. Lameness (grade 1/5) was evident in the right thoracic limb with pain on palpation and manipulation of the caudal aspect of the distal phalangeal and elbow joints. The lameness resolved after injection of lidocaine (Lidocaine 2%, VetOne a ) (2,000 U) into the elbow joint. Joint fluid analysis and radiographs of the distal phalangeal and elbow joints were considered normal. Spinal radiographs, evaluated because of rear limb weakness, also were considered normal. Radiographs of the thorax disclosed ventral alveolar opacities, consistent with aspiration pneumonia. The foal was somnolent on neurological examination and in sternal recumbency, but when assisted it/he could stand and walk. The lower lip drooped bilaterally and the palpebral fissures were decreased bilaterally because of upper eyelid ptosis. Normal palpebral reflexes were

Objective—To establish an in vivo method for matrix metalloproteinase (MMP)-2 and MMP-9 induction in horses via IV administration of lipopolysaccharide (LPS) and to evaluate the ability of doxycycline, oxytetracycline, flunixin meglumine, and pentoxifylline to inhibit equine MMP-2 and MMP-9 production. Animals—29 adult horses of various ages and breeds and either sex. Procedures—In part 1, horses received an IV administration of LPS (n = 5) or saline (0.9% NaCl) solution (5). Venous blood samples were collected before and at specified times for 24 hours after infusion. Plasma was harvested and analyzed for MMP-2 and MMP-9 activities via zymography. In part 2, horses received doxycycline (n = 5), oxytetracycline (5), flunixin meglumine (5), or pentoxifylline (4) before and for up to 12 hours after administration of LPS. Plasma was obtained and analyzed, and results were compared with results from the LPS-infused horses of part 1. Results—Administration of LPS significantly increased MMP-2 and MMP-9 activities in the venous circulation of horses. All MMP inhibitors significantly decreased LPS-induced increases in MMP activities but to differing degrees. Pentoxifylline and oxytetracycline appeared to be the most effective MMP-2 and MMP-9 inhibitors, whereas doxycycline and flunixin meglumine were more effective at inhibiting MMP-2 activity than MMP-9 activity. Conclusions and Clinical Relevance—IV administration of LPS to horses caused increased venous plasma activities of MMP-2 and MMP-9. These MMP activities were reduced by pentoxifylline and oxytetracycline, suggesting that further evaluation of these medications for treatment and prevention of MMP-associated diseases in horses is indicated.

All cases of laminitis are characterized by failure of the attachment of the epidermal cells of the epidermal laminae to the underlying basement membrane of the dermal laminae despite the diversity of diseases that underlie the syndrome. The preponderance of evidence supports roles for inflammation, metabolic derangement, endothelial and venous dysfunction, and matrix degradation as causes of laminitis. Inflammation, oxidant stress, and matrix degradation may be factors common to each of these mechanisms that lead to the laminar damage of laminitis. The understanding of the pathophysiology and progression of the disease is incomplete, and this limits efforts to prevent and treat this devastating disease successfully. However, scientific investigations are occurring at a phenomenal rate and shedding light on the pathophysiologic events involved with laminitis.

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.

Objective—To quantify changes in endothelium-derived factors and relate those changes to various aspects of digital hemodynamics during the prodromal stages of carbohydrate overload (CHO)-induced laminitis in horses. Animals—20 adult horses without abnormalities of the digit. Procedures—Digital and jugular venous blood samples were collected at 1-hour intervals (for assessment of endothelin-1 [ET-1] immunoreactivity and measurement of glucose, insulin, and nitric oxide [NO] concentrations) or 4-hour intervals (CBC and platelet-neutrophil aggregate assessment) for 8 hours or 16 hours after induction of CHO-associated laminitis in horses treated with an ET-1 antagonist. Effects of treatment, collection site, and time and the random effects of horse on each variable were analyzed by use of a repeated-measures model. Where treatment and collection site had no significant effect, data were combined. Results—Compared with baseline values, CHO resulted in changes in several variables, including a significant increase from baseline in digital blood ET-like immunoreactivity at 11 hours; digital blood ET-like immunoreactivity was significantly greater than that in jugular venous blood at 8, 9, 11, and 12 hours. Digital and jugular venous blood concentrations of glucose increased from baseline significantly at 3, 4, and 5 hours; insulin concentration increased significantly at 5 hours; and the number of platelet-neutrophil aggregates increased significantly at 12 hours. Conclusions and Clinical Relevance—In horses, concurrent increases in venous blood ET-1 immunoreactivity, insulin and glucose concentrations, and platelet-neutrophil aggregates support a role of endothelial dysfunction in the pathogenesis of CHO-induced laminitis.

Objective—To characterize the in vitro response of equine cecal longitudinal smooth muscle (CLSM) to endothelin (ET)-1 and assess the role of ETA and ETB receptors in those ET-1–induced responses. Animals—36 horses without gastrointestinal tract disease. Procedure—To determine cumulative concentrationresponse relationships, CLSM strips were suspended in tissue baths containing graded concentrations of ET-1 (10–9 to 10–6M) with or without BQ-123 (ETA receptor antagonist); with or without IRL-1038 (ETB receptor antagonist); or with both antagonists at concentrations of 10–9, 10–7, and 10–5M. To determine the percentage change in baseline tension of CLSM, the areas under the curve during the 3-minute periods before and after addition of each dose were compared . Also, the effects of ET-1 and a combination of selective ETA and ETB receptor antagonists on electrically evoked contractions were studied. Results—ET-1 caused sustained increases in CLSM tension in a concentration-dependent manner. Contractile responses to ET-1 were not significantly inhibited by either BQ-123 or IRL-1038 alone at any concentration; however, responses were significantly inhibited by exposure to the antagonists together at a concentration of 10–5M. Electrical field stimulation did not change the spontaneous contractile activity of CLSM and did not significantly alter the tissue response to ET-1, BQ-123, or IRL-1038. Conclusions and Clinical Relevance—Results indicated that ET-1 has a contractile effect on equine CLSM that is mediated via ETA and ETB receptors. In vitro spontaneous contractions of equine CLSM apparently originate in the smooth muscle and not the enteric nervous system. (Am J Vet Res 2005;66:1202–1208)

Objective—To determine and compare the number, type, location, and distribution of apoptotic epidermal cells in the laminae of clinically normal horses and horses with laminitis. Sample Population—Formalin-fixed samples of digital lamellar tissue from 47 horses (including clinically normal horses [controls; n = 7], horses with acute [4] and chronic [7] naturally acquired laminitis, and horses with black walnut extract-induced [11] or carbohydrate overload-induced [18] laminitis). Procedure—Blocks of paraffin-embedded lamellar tissues were stained for DNA fragmentation with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Differential immunohistochemical staining for caspases 3 and 14 were used to confirm apoptosis. Results—The number of TUNEL-positive epidermal cells per 0.1 mm of primary laminae was significantly greater in the acute laminitis group than in the other groups. In the acute laminitis group, there were 17 and 1,025 times as many TUNEL-positive basal layer cells and keratinocytes, respectively, compared with the control group. Apoptosis of TUNEL-positive basal layer cells was confirmed by results of caspase 3 immunohistochemical staining. The TUNEL-positive keratinocytes did not stain for caspases 3 or 14. Conclusions and Clinical Relevance—The large number of apoptotic basal layer cells detected in the lamellar tissue of horses with acute naturally acquired laminitis suggests that apoptosis may be important in the development of acute laminitis. The role of the large number of TUNEL-positive keratinocytes detected in the interface of primary and secondary epidermal laminae of horses with acute laminitis remains to be elucidated. ( Am J Vet Res 2004;65:578–585)

Objective—To evaluate systemic effects of IV infusion of ATP-MgCl2 subsequent to infusion of a low dose of endotoxin in horses. Animals—12 adult horses. Procedure—Horses were administered endotoxin (lipopolysaccharide [LPS]) or saline (0.9% NaCl) solution, IV, during a 30-minute period. Immediately thereafter, horses in each group were infused IV with ATP-MgCl2 or saline solution. Two weeks later, horses were administered the opposite solution (LPS or saline solution), but it was followed by the same infusion as 2 weeks previously (ie, ATP-MgCl2 or saline solution). Cardiopulmonary and clinicopathologic variables, cytokine activity, and endothelin (ET) concentrations were recorded. Results—IV infusion of ATP-MgCl2 after administration of a low dose of endotoxin failed to attenuate the cardiopulmonary, clinicopathologic, and cytokine alterations that develop secondary to endotoxin exposure. The combination of LPS and ATP-MgCl2 potentiated pulmonary hypertension, leukopenia, and neutropenia when compared with the combination of LPS and saline solution. The combination of LPS and ATP-MgCl2 resulted in thrombocytopenia. Endothelin concentration was increased in jugular venous and pulmonary arterial plasma in horses receiving LPS and ATP-MgCl2. Similar increases were not observed with LPS and saline solution. Conclusions and Clinical Relevance—Administration of ATP-MgCl2 did not protect horses from systemic effects of experimentally induced endotoxemia. Furthermore, the use of ATP-MgCl2 during endotoxemia may worsen the cardiopulmonary and clinicopathologic status of affected horses. Because ATP and other adenine nucleotides are released from cells during shock, their potential role in the development of hemodynamic derangements, leukocyte adherence, and coagulopathies during endotoxemic episodes warrants further investigation. (Am J Vet Res 2004;65: 225–237)

Objective—To characterize alterations in systemic and local colonic hemodynamic variables associated with IV infusion of ATP-MgCl2 in healthy anesthetized horses. Animals—12 adult horses. Procedure—Six horses were given ATP-MgCl2, IV, beginning at a rate of 0.1 mg of ATP/kg of body weight/min with incremental increases until a rate of 1.0 mg/kg/min was achieved. The remaining 6 horses were given an equivalent volume of saline (0.9% NaCl) solution over the same time period. Colonic and systemic hemodynamic variables and colonic plasma nitric oxide (NO) concentrations were determined before, during, and after infusion. Results—Infusion of ATP-MgCl2 caused a rate-dependent decrease in systemic and colonic vascular resistance, principally via its vasodilatory effects. A rate of 0.3 mg of ATP/kg/min caused a significant decrease in systemic and colonic arterial pressure and colonic vascular resistance without a significant corresponding decrease in colonic arterial blood flow. Consistent alterations in NO concentrations of plasma obtained from colonic vasculature were not detected, despite profound vasodilatation of the colonic arterial vasculature. Conclusions and Clinical Relevance—Results revealed that IV infusion of ATP-MgCl2 may be beneficial in maintaining colonic perfusion in horses with ischemia of the gastrointestinal tract, provided a sufficient pressure gradient exists to maintain blood flow. (Am J Vet Res 2001;62:1240–1249)

We describe the echocardiographic findings in a 4-day-old thoroughbred foal with an aortico-pulmonary septal defect. The foal had labored breathing, cyanotic mucous membranes and a continuous grade 5/6 heart murmur with point of maximal intensity over the base of the heart on the right side. Echocardiographically, there was a large communication between the aorta and the pulmonary artery just dorsal to the base of the heart. The cardiac anomaly seen during the echocardiographic exam was confirmed at necropsy where a large communication between the two great vessels was observed. These findings correlate with previous studies in humans, dogs, and cats. The possible failure in the embryologic development that led to this unusual cardiac anomaly is discussed.

Hyperinsulinemia causes laminitis experimentally and is a risk factor for naturally occurring laminitis. The aim of this study was to investigate the effects of insulin on laminar vascular relaxation and to induce insulin-associated vascular dysfunction in vitro. Relaxation responses of isolated laminar arterial and venous rings to acetylcholine and insulin were evaluated. To alter vascular function in response to insulin, all vessel rings were incubated with insulin or vehicle, submaximally contracted, administered insulin again and relaxation responses recorded. Laminar arteries were also incubated with the mitogen-activated protein kinase (MAPK) inhibitor, PD-98059. Relaxation in response to acetylcholine was not different between arteries and veins, but veins relaxed less in response to insulin than arteries. In arteries incubated with insulin, the subsequent relaxation response to insulin was blunted. Veins had minimal relaxation to insulin regardless of incubation. Arteries incubated with PD-98059 relaxed more in response to insulin than arteries not exposed to PD-98059, indicating that MAPK plays a role in maintenance of basal tone in laminar arteries. A differing response of laminar veins and arteries to insulin-induced relaxation may be important in understanding the link between hyperinsulinemia and laminitis. In vitro induction of vascular dysfunction in response to insulin in laminar arteries may be useful for testing therapeutic interventions and for understanding the pathophysiology of laminitis.

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 β-catenin and T-cell Factor 4, consistent with regulation by the canonical Wnt signaling pathway. In addition, gene and protein expression of β-catenin correlates positively with that of integrin β4 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 β catenin and integrin β4 in laminar basal epithelial cells reduces cell:cell and cell:basement membrane attachment, thus, destabilizing the laminar epidermal:dermal junction.

Objective—To determine the effects of clenbuterol, at a dosage of up to 3.2 μg/kg for 14 days, PO, on skeletal and cardiac muscle in healthy horses undergoing treadmill exercise. Animals—12 healthy horses from 3 to 10 years old. Procedures—Horses were randomly assigned to a control group (n = 6) or clenbuterol group (6) and received either saline (0.9% NaCl) solution or clenbuterol, PO, every 12 hours for 14 days. Horses were subjected to submaximal treadmill exercise daily during treatment. Muscle biopsy specimens were collected before and after treatment for determination of apoptosis. Echocardiographic measurements, serum clenbuterol and cardiac troponin I concentrations, and serum activities of creatine kinase and aspartate aminotransferase were measured before, during, and after treatment. Jugular venous blood samples were collected every 3 days during treatment. Echocardiography was repeated every 7 days after beginning treatment. Response variables were compared between treatment groups and across time periods. Results—No significant effect of clenbuterol or exercise on response variables was found between treatment and control groups at any time point or within groups over time. Conclusions and Clinical Relevance—Results did not reveal any adverse effects of treatment with an approved dose of clenbuterol on equine cardiac or skeletal muscle in the small number of horses tested.

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.

A 5-day-old Thoroughbred colt was presented with profuse watery diarrhea, hypovolemic shock, and a patent urachus. Despite intensive medical therapy, the colt was euthanized 15 d later due to poor clinical response. Necropsy revealed a small intestinal structural abnormality that formed a closed jejunal ring. Although rare, intestinal malformations should be considered in neonatal foals with clinical signs resembling enteritis.

The role of arachidonic acid metabolites in the forestomach stasis induced by Escherichia coli endotoxin was evaluated. Six adult Holstein cows received saline solution; endotoxin at 1, 10, and 100 ng/kg of body weight; flunixin meglumine at 1.1 mg/kg of body weight; and flunixin meglumine at 1.1 mg/kg plus endotoxin at 100 ng/kg. The frequency of reticulorumen contractions, mental attitude, body temperature, respiratory rate, heart rate, and plasma concentration of prostaglandin E2, prostacyclin, and thromboxane were evaluated. Administration of saline solution and endotoxin at 1 ng/kg had no significant effects. Administration of endotoxin at 10 ng/kg did not cause significant clinical effects or alter reticulorumen contractions but enhanced synthesis of thromboxane. Administration of endotoxin at 100 ng/kg caused mild clinical signs of stasis, reduced the frequency of reticulorumen contractions, and enhanced synthesis of thromboxane and prostacyclin. Reticulorumen stasis was not accompanied by an increase in the plasma concentration of prostaglandin E2. Flunixin meglumine abolished endotoxin-induced reticulorumen stasis, tachycardia, and synthesis of arachidonic acid metabolites. Reticulorumen stasis during bovine endotoxemia is caused either by enhanced synthesis of an arachidonic acid metabolite other than prostaglandin E2 or by local synthesis of prostaglandin E2.

Background: Summer pasture-associated recurrent airway obstruction (SPA-RAO), a seasonal airway obstructive disease of horses, is characterized by clinical exacerbation after exposure to pasture during warm months of the year. Endothelin (ET)-1, potent bronchoconstrictor, mitogen, secretagogue, and proinflammatory mediator, has been implicated in the pathogenesis of asthma and equine heaves. Hypothesis: Immunoreactive ET-1 concentrations increase during clinical exacerbation and return to basal values during periods of disease remission. Animals: Twelve horses, 6 affected with SPA-RAO and 6 nonaffected. Methods: Prospective, observational study. Bronchoalveolar lavage fluid (BALF), arterial and venous plasma samples, and clinical variables were obtained from affected horses during clinical exacerbation and remission. Samples and data of nonaffected horses were collected during the summer and winter on dates similar to affected horses. Immunoreactive ET-1 was determined using a commercial ELISA. Results: The median and range ET-1 concentrations (pg/ml) in arterial (1.3, 0.7–1.8) and venous (1.3, 1.2–1.7) plasma and in BALF (0.3, 0.2–0.4), and pulmonary epithelial lining fluid (PELF) (25.5, 21–50) were greater in affected horses during clinical exacerbation compared with remission (P < .01). The concentrations of immunoreactive ET-1 were greater in affected horses during clinical exacerbation compared with nonaffected horses (P < .05). Conclusions and Clinical Importance: During clinical exacerbation of SPA-RAO, ET-1 is increased in circulation and pulmonary secretions. Intervention with ET receptor antagonists should provide further information on the role of ET-1 in SPA-RAO.

To determine the magnitude and duration of effects of acepromazine administered intramuscularly (IM) on digital and systemic hemodynamic variables in clinically healthy horses.Experimental study.Healthy adult horses (n=12). Methods- An ultrasonic Doppler flow probe was surgically implanted around the medial palmar digital artery before the study. Catheters were inserted in the transverse facial artery, lateral palmar digital artery, and jugular vein. A treatment group (n=6) was administered 0.04 mg/kg body weight of acepromazine IM; control horses (n=6) were administered an equivalent volume of saline IM. Palmar digital blood flow, and digital and facial arterial pressures were measured at baseline and for 6 hours after administration. Venous blood was collected for measurement of packed cell volume (PCV).Horses administered acepromazine had significantly lower facial arterial pressure compared with control horses administered saline. Palmar digital arterial blood flow in acepromazine-treated horses was not significantly different from that in control horses but increased significantly post-administration, compared with the respective baseline values for acepromazine-treated horses. PCV significantly decreased in horses administered acepromazine compared with their respective baseline value.IM acepromazine causes hypotension and increases palmar digital blood flow over time but the magnitude of the effect on digital blood flow was not sufficient to yield differences compared with saline-treated horses.IM acepromazine has a modest effect on palmar digital blood flow, facial arterial pressures and PCV in healthy horses with minimal sedation.

The goals of this study were to determine the concentration-response (C-R) relationship of endothelin-1 (ET-1), compare 2 ET-receptor antagonists and determine the antagonist concentrations that block the vasomotor effects of ET-1, and compare the effectiveness of ET-1 and previously studied vasoconstrictors in equine palmar digital arterial and venous rings in vitro. Vessel rings from 8 nonlaminitic horses were placed in Tyrode's solution, 1 side fixed to the floor of an organ bath and the other side fixed to a force-displacement transducer. Two separate studies were conducted: (I) incubation with a single ET-receptor antagonist (PD142893 or PD145065 at a concentration of 10(-7), 10(-6), or 10(-5) M), followed by determination of an ET-1 C-R curve (using concentrations of 10(-10) to 10(-6) M) for medial vessel rings; and (II) comparison of ET-1 with norepinephrine and histamine (10(-10) to 10(-6) M) and comparison of contractile responses of medial and lateral vessel rings. In study I, ET-1 administration caused pronounced and sustained concentration-dependent contraction of vessel rings; these contractile responses were decreased by 10(-5) M PD142893 and were completely blocked by 10(-5) M PD145065. Venous rings had greater apparent maximum contraction in response to ET-1 than arterial rings. In study II, the relative sensitivity of norepinephrine was found to be equivalent to that of ET-1, whereas that of histamine was lower. No significant differences were observed between responses of medial versus lateral vessel rings. Thus, ET-1 is a potent vasoconstrictor of equine palmar digital arteries and veins, and the ET-receptor antagonist PD145065 is more effective than PD142893 in inhibiting these contractile effects in vitro.

Objective—To evaluate changes in digital vascular function in horses with carbohydrate overload (CHO)-induced laminitis and determine the effects of an endothelin (ET) receptor antagonist and nitroglycerin on laminitis-associated vascular dysfunction. Animals—20 adult horses without abnormalities of the digit. Procedures—Hemodynamic variables were recorded before (baseline) and hourly after all horses were administered a CHO ration via nasogastric tube. In 4 groups of 5 horses each, saline (0.9% NaCl) solution or ET receptor antagonist (10−5M in digital blood) was administered into the digital arterial circulation according to 1 of 2 schedules. During anesthesia, blood flow; arterial, venous, and capillary pressures; and total, precapillary, and postcapillary resistances were measured in an isolated perfused digit of each horse. In all groups, nitroglycerin was infused (10−5M in digital blood), and digital microvascular assessments were repeated. Results—The CHO caused a significant decrease in right atrial pressure by 14 hours that was not affected by administration of saline solution or ET receptor antagonist. In isolated digits of anesthetized horses, CHO resulted in a significant decrease in digital blood flow associated with a significant increase in total and postcapillary resistances. Treatment with the ET receptor antagonist and nitroglycerin caused a significant decrease in total resistance. Postcapillary resistance was significantly decreased following treatment with the ET receptor antagonist but was not altered by treatment with nitroglycerin. Conclusions and Clinical Relevance—Treatment with an ET receptor antagonist and nitroglycerin resulted in significant improvement in vascular resistance in isolated perfused digits of anesthetized horses with CHO-induced laminitis.

Objective— To measure and compare palmar digital venous plasma nitric oxide (NO) concentrations and digital arterial blood flow after application of topical nitroglycerine (NTG). Study Design— Experimental study. Animals— Healthy adult horses (n=8). Methods— Digital blood flow was measured by an ultrasonic Doppler flow probe surgically implanted around the medial palmar digital artery. Blood was collected from a catheter placed in the medial palmar digital vein for quantification of NO. NTG patches, NTG ointment or control patches were placed over the palmar digital vessels at the level of the fetlock. Two horses had an intra-arterial infusion of an NTG solution into the medial palmar digital artery in a pilot study. Results— Digital arterial blood flow did not change significantly with application of the NTG patches, NTG ointment, or control patches. There were no statistically significant or biologically important changes in digital venous NO concentrations across time or between treated and control horses. In the pilot study, digital arterial blood flow and palmar digital venous NO concentrations increased with intra-arterial infusion of NTG. Conclusions— In clinically healthy horses, digital arterial blood flow and digital venous plasma NO concentrations did not change significantly with application of the NTG patches/ointment. These treatments are unlikely to have an effect on the digital vasculature of laminitic horses, however, further investigation is warranted. Clinical Relevance— Although NTG patches have been used as a method of decreasing vasomotor tone and improving digital blood flow in horses with laminitis, this study provides evidence in healthy conscious horses that this treatment is not effective in altering digital blood flow.

Objective—To characterize the in vitro response of circular and longitudinal myometrial layers of the uterine horn (CMLH and LMLH, respectively) of horses to endothelin (ET)-1 by use of specific ETA (BQ-123) and ETB (IRL-1038) receptor antagonists. Sample Population—Uteruses from 10 nongravid mares in anestrus. Procedure—Muscle strips from the CMLH and LMLH were suspended in tissue baths and connected to force-displacement transducers interfaced with a polygraph. Strips were incubated for 45-minute intervals with no antagonist (control specimens), and 3 concentrations (10–9, 10–7, and 10–5M) of BQ-123, IRL- 1038, or BQ-123 and IRL-1038 before concentrationresponse curves to ET-1 were generated. Contractile response to cumulative concentrations of ET-1 (10–9 to 10–6M) was quantified by measuring change in the area under the curve (AUC) for the 3-minute period after each ET-1 dose. Results—ET-1 caused concentration-dependent contraction of the CMLH and LMLH specimens. Application of BQ-123 decreased AUC values for both layers. Application of IRL-1038 increased the AUC value for LMLH specimens but did not affect the CMLH value. The combination of BQ-123 and IRL-1038 decreased the AUC value for LMLH tissue and increased that for CMLH tissue. Conclusions and Clinical Relevance—ET-1 causes contraction of the CMLH and LMLH in nongravid horses. In both layers, ETA receptors mediate contraction but the role of ETB receptors remains unclear. In the LMLH, ETA receptors have a dominant role; the presence of another receptor or receptor subtype within this layer is suggested. These findings support a physiologic role for ET-1 in uterine contractility. (Am J Vet Res 2005;66:1094–1100)

Objective—To examine the secretory response (in the presence and absence of prostaglandin inhibition) in vitro and structural alterations of colonic mucosa in horses after intragastric administration of black walnut extract (BWE). Animals—14 adult horses. Procedure—Seven horses were administered BWE intragastrically and monitored for 11 hours. Tissue samples were obtained from the right ventral, left ventral, and right dorsal colons (RVC, LVC, and RDC, respectively) of the 7 BWE-treated and 7 control horses. Tissue samples were examined via light microscopy, and the extent of hemorrhage, edema, and granulocytic cellular infiltration (neutrophils and eosinophils) was graded. Colonic mucosal segments were incubated with or without flunixin meglumine (FLM) for 240 minutes; spontaneous electrical potential difference and short-circuit current (Isc) were recorded and used to calculate mucosal resistance. Results—Colonic tissues from BWE-treated horses (with or without FLM exposure) had an overall greater Isc during the 240-minute incubation period, compared with tissues from control horses. The resistance pattern in RVC, LVC, and RDC samples (with or without FLM exposure) from BWE-treated horses was decreased overall, compared with control tissues (with or without FLM exposure). Histologically, colonic mucosal tissues from BWE-treated horses had more severe inflammation (involving primarily eosinophils), edema, and hemorrhage, compared with tissue from control horses. Conclusions and Clinical Relevance—In horses, BWE administration appears to cause an inflammatory response in colonic mucosal epithelium that results in mucosal barrier compromise as indicated by decreased mucosal resistance with presumed concomitant electrogenic chloride secretory response, which is not associated with prostaglandin mediation. (Am J Vet Res 2005;66:443–449)

AL amyloidosis is the most common type of systemic amyloidosis in humans, and it is frequently associated with multiple myeloma. But, AL amyloidosis is very rare in domestic animals. A 16-year-old Quarter horse gelding was diagnosed with systemic AL amyloidosis associated with multiple myeloma. Clinical problems were rapid weight loss, muscle atrophy, soft unformed stool, and ventral edema. Grossly, diffuse gastrointestinal hemorrhage, markedly thickened jejunal mucosa, and splenomegaly were present. Microscopically, diffuse severe amyloid deposits were present in the lamina propria of glandular stomach, duodenum, and jejunum. Much of the spleen and sternal bone marrow was replaced by neoplastic round cells, and multiple foci of amyloid were also present in the spleen and bone marrow. Electron microscopy revealed the neoplastic round cells to be of plasma cell origin, and the amyloid showed a strongly positive immunoreactivity with polyclonal anti-human immunoglobin lambda light-chain antisera. To our knowledge, this is the second report describing systemic AL amyloidosis in domestic animals-associated plasma cell neoplasia and the first associated with multiple myeloma, as is common in humans.

Summary Reasons for performing study: Abdominal insufflation is performed routinely during laparoscopy in horses to improve visualisation and facilitate instrument and visceral manipulations during surgery. It has been shown that high-pressure pneumoperitoneum with carbon dioxide (CO2) has deleterious cardiopulmonary effects in dorsally recumbent, mechanically ventilated, halothane-anaesthetised horses. There is no information on the effects of CO2 pneumoperitoneum on cardiopulmonary function and haematology, plasma chemistry and peritoneal fluid (PF) variables in standing sedated horses during laparoscopic surgery. Objectives: To determine the effects of high pressure CO2 pneumoperitoneum in standing sedated horses on cardiopulmonary function, blood gas, haematology, plasma chemistry and PF variables. Methods: Six healthy, mature horses were sedated with an i.v. bolus of detomidine (0.02 mg/kg bwt) and butorphanol (0.02 mg/kg bwt) and instrumented to determine the changes in cardiopulmonary function, haematology, serum chemistry and PF values during and after pneumoperitoneum with CO2 to 15 mmHg pressure for standing laparoscopy. Each horse was assigned at random to either a standing left flank exploratory laparoscopy (LFL) with CO2 pneumoperitoneum or sham procedure (SLFL) without insufflation, and instrumented for measurement of cardiopulmonary variables. Each horse underwent a second procedure in crossover fashion one month later so that all 6 horses had both an LFL and SLFL performed. Cardiopulmonary variables and blood gas analyses were obtained 5 mins after sedation and every 15 mins during 60 mins baseline (BL), insufflation (15 mmHg) and desufflation. Haematology, serum chemistry analysis and PF analysis were performed at BL, insufflation and desufflation, and 24 h after the conclusion of each procedure. Results: Significant decreases in heart rate, cardiac output and cardiac index and significant increases in mean right atrial pressure, systemic vascular resistance and pulmonary vascular resistance were recorded immediately after and during sedation in both groups of horses. Pneumoperitoneum with CO2 at 15 mmHg had no significant effect on cardiopulmonary function during surgery. There were no significant differences in blood gas, haematology or plasma chemistry values within or between groups at any time interval during the study. There was a significant increase in the PF total nucleated cell count 24 h following LFL compared to baseline values for LFL or SLFL at 24 h. There were no differences in PF protein concentrations within or between groups at any time interval. Conclusions: Pneumoperitoneum with CO2 during standing laparoscopy in healthy horses does not cause adverse alterations in cardiopulmonary, haematology or plasma chemistry variables, but does induce a mild inflammatory response within the peritoneal cavity. Potential relevance: High pressure (15 mmHg) pneumoperitoneum in standing sedated mature horses for laparoscopic surgery can be performed safely without any short-term or cumulative adverse effects on haemodynamic or cardiopulmonary function.

Objective—To compare effects of oxytocin, acepromazine maleate, xylazine hydrochloride-butorphanol tartrate, guaifenesin, and detomidine hydrochloride on esophageal manometric pressure in horses. Animals—8 healthy adult horses. Procedure—A nasogastric tube, modified with 3 polyethylene tubes that exited at the postpharyngeal area, thoracic inlet, and distal portion of the esophagus, was fitted for each horse. Amplitude, duration, and rate of propagation of pressure waveforms induced by swallows were measured at 5, 10, 20, 30, and 40 minutes after administration of oxytocin, detomidine, acepromazine, xylazine-butorphanol, guaifenesin, or saline (0.9% NaCl) solution. Number of spontaneous swallows, spontaneous events (contractions that occurred in the absence of a swallow stimulus), and high-pressure events (sustained increases in baseline pressure of > 10 mm Hg) were compared before and after drug administration. Results—At 5 minutes after administration, detomidine increased waveform amplitude and decreased waveform duration at the thoracic inlet. At 10 minutes after administration, detomidine increased waveform duration at the thoracic inlet. Acepromazine administration increased the number of spontaneous events at the thoracic inlet and distal portion of the esophagus. Acepromazine and detomidine administration increased the number of high-pressure events at the thoracic inlet. Guaifenesin administration increased the number of spontaneous events at the thoracic inlet. Xylazine-butorphanol, detomidine, acepromazine, and guaifenesin administration decreased the number of spontaneous swallows. Conclusions and Clinical Relevance—Detomidine, acepromazine, and a combination of xylazine butorphanol had the greatest effect on esophageal motility when evaluated manometrically. Reduction in spontaneous swallowing and changes in normal, coordinated peristaltic activity are the most clinically relevant effects. (Am J Vet Res 2002;63:1738–1744)

Objective—To characterize the in vitro effects of oxytocin, acepromazine, xylazine, butorphanol, detomidine, dantrolene, isoproterenol, and terbutaline on skeletal and smooth muscle from the equine esophagus. Animals—14 adult horses without digestive tract disease. Procedure—Circular and longitudinal strips from the skeletal and smooth muscle of the esophagus were suspended in tissue baths, connected to force-displacement transducers interfaced with a physiograph, and electrical field stimulation was applied. Cumulative concentration-response curves were generated for oxytocin, acepromazine, xylazine, detomidine, butorphanol, isoproterenol, terbutaline, and dantrolene. Mean maximum twitch amplitude for 3 contractions/min was recorded and compared with predrug-vehicle values for the skeletal muscle segments, and area under the curve (AUC) for 3 contractions/min was compared with predrug-vehicle values for the smooth muscle segments. Results—No drugs caused a significant change in skeletal muscle response. In smooth muscle, isoproterenol, terbutaline, and oxytocin significantly reduced AUC in a concentration-dependent manner. Maximum reduction in AUC was 69% at 10–4M for isoproterenol, 63% at 10–5M for terbutaline, and 64% at 10–4M for oxytocin. Conclusions and Clinical Relevance—Isoproterenol, terbutaline, and oxytocin cause relaxation of the smooth muscle portion of the esophagus. The clinical relaxant effects on the proximal portion of the esophagus reported of drugs such as oxytocin, detomidine, and acepromazine may be the result of centrally mediated mechanisms. (Am J Vet Res 2002;63:1732–1737)

Objective—To compare plasma endothelin (ET)- like immunoreactivity between healthy horses and those with naturally acquired gastrointestinal tract disorders. Animals—29 healthy horses and 142 horses with gastrointestinal tract disorders. Procedure—Blood samples were collected from healthy horses and from horses with gastrointestinal tract disorders prior to treatment. Magnitude and duration of abnormal clinical signs were recorded, and clinical variables were assessed via thorough physical examinations. Plasma concentrations of ET-like immunoreactivity were measured by use of a radioimmunoassay for human endothelin-1, and CBC and plasma biochemical analyses were performed. Results—Plasma ET-like immunoreactivity concentration was significantly increased in horses with gastrointestinal tract disorders, compared with healthy horses. Median plasma concentration of ET-like immunoreactivity was 1.80 pg/ml (range, 1.09 to 3.2 pg/ml) in healthy horses. Plasma ET-like immunoreactivity was greatest in horses with strangulating largeintestinal obstruction (median, 10.02 pg/ml; range, 3.8 to 22.62 pg/ml), peritonitis (9.19 pg/ml; 7.89 to 25.83 pg/ml), and enterocolitis (8.89 pg/ml; 6.30 to 18.36 pg/ml). Concentration of ET-like immunoreactivity was significantly associated with survival, PCV, and duration of signs of pain. However, correlations for associations with PCV and duration of pain were low. Conclusions and Clinical Relevance—Horses with gastrointestinal tract disorders have increased plasma concentrations of ET-like immunoreactivity, compared with healthy horses. The greatest values were detected in horses with large-intestinal strangulating obstructions, peritonitis, and enterocolitis. This suggests a potential involvement of ET in the pathogenesis of certain gastrointestinal tract disorders in horses. (Am J Vet Res 2002;63:454–458)

The present study investigated the possibility that nitric oxide is a nonadrenergic, noncholinergic neurotransmitter of nerves that are intrinsic to the forestomach. Tunica muscularis, myenteric plexus preparations of bovine reticulum and rumen were maintained in vitro in a physiological solution of buffer that contained scopolamine. Trains of electric field stimulation transiently reduced (relaxed) the tone induced by BaCl2. NG-Nitro-L-arginine methyl ester, a nitric oxide synthase competitive antagonist, inhibited relaxation of the rumen and reticulum preparations that had been induced by the electrical field. The actions of NG-nitro-L-arginine methyl ester were partially reversed by L-arginine. These data suggest that nitric oxide, or a related substance, is an inhibitory neurotransmitter of nerves that are intrinsic to tunica muscularis, myenteric plexus preparations of the bovine forestomach.

The objective of this study was to determine whether blockade of alpha(-2) adrenergic receptors would restore reticulorumen motility during toxaemia in cows. Reticulorumen contractions were measured via a water-filled balloon connected to a pressure transducer. Intravenous infusion of endotoxin (100 ng kg-1 over 30 min) significantly decreased the number of reticulorumen contractions. Intravenous infusion of yohimbine (125 micrograms kg-1 over 30 min) alone did not affect reticulorumen contractions. However, when yohimbine (125 micrograms kg-1 over 30 min) was infused concurrently with endotoxin (100 ng kg-1 over 30 min), the effects of endotoxin on reticulorumen contraction frequency decreased, suggesting that endotoxaemia causes reticulorumen stasis via a mechanism that involves alpha(-2) adrenergic receptors.