Catherine Delesalle

Risk Factors for Equine Postoperative Ileus and Effectiveness of Prophylactic Lidocaine

BACKGROUND Postoperative ileus (POI) is a frequent and often fatal complication of colic surgery. Reliably effective treatments are not available. OBJECTIVES To determine risk factors and protective factors associated with POI, and to assess the effect of lidocaine IV on short-term survival. ANIMALS One hundred and twenty-six horses that underwent small intestinal colic surgery and that survived for at least 24 hours postoperatively. METHODS Retrospective cross-sectional study. The association of 31 pre-, intra-, and postoperative variables with POI and the association of lidocaine treatment with short-term survival were investigated. Associations were evaluated with univariable logistic regression models, followed by multivariable analysis. RESULTS Significant associations of high heart rate (odds ratio [OR] = 1.05, 95% confidence interval [CI] 1.03-1.08), the presence of more than 8 L of reflux at admission (OR = 3.02, 95% CI 1.13-8.02) and the performance of a small intestinal resection (OR = 2.46, 95% CI 1.15-5.27) with an increased probability of POI were demonstrated. Prophylactic lidocaine treatment was significantly associated with a reduced incidence of POI (OR = 0.25, 95% CI 0.11-0.56). Lidocaine treatment was also significantly associated with enhanced short-term survival (OR = 0.30, 95% CI 0.09-0.98). CONCLUSIONS AND CLINICAL IMPORTANCE The variables associated with an increased risk of POI can be useful in identifying horses at risk of POI and in providing a more accurate prognosis. The results are supportive for lidocaine IV as an effective prokinetic treatment after small intestinal colic surgery.

Use of plasma ionized calcium levels and Ca2+ substitution response patterns as prognostic parameters for ileus and survival in colic horses.

Summary Objective: Hypocalcaemia is a common finding in horses with enterocolitis and severe gastrointestinal disorders. The aims of this study were to investigate in colic horses !)the parameters related to hypocalcaemia, 2)the influence of hypocalcaemia on outcome and 3)the possible beneficial effect of Ca2+ substitution. Design: Randomized controlled trial. Setting: Intensive care unit. Patients: One hundred forty‐four horses that were admitted with an acute abdomen during a 1.5 year period were enrolled and daily evaluated for clinical criteria and whole blood ionized Ca levels. Colic horses with hypocalcaemia were randomly assigned to receive Ca2+. Interventions: Analysis of heparinised whole blood samples. Horses that were assigned to be treated received 400 mEq Ca2+ diluted in 10L of Ringers lactate solution every 24 h until low reference range limits were obtained or until death. Measurements and main results: 88% of all colic patients showed blood ionized Ca levels below the reference range at the time of admission. Multivariable analysis revealed that the presence of reflux signs of endotoxaemia increased Packed Cell Volume (PCV) alkalinization of pH and the interaction PCV/pH all predispose colic horses to low ionized Ca2+ levels at the time of admission. The Odds for developing ileus during hospitalization are ± 11.94 times larger for horses in the “very low” calcaemia interval in comparison with normocalcaemic horses. The Odds for fatal outcome are respectively ± 9.82 and 8.33 times larger for horses in the “very low” and “low” calcaemia interval. Ca2+ substitution increased the probability of survival provided that Ca2+ levels could be normalized. The lack of an upward calcaemia response despite repetitive Ca2+ substitutions can be guarded as a poor ominous sign. Conclusions: Hypocalcaemia in colic horses is of prognostic relevance both with regard to survival as to the probability of development of ileus during hospitalization. This study shows the importance of routine measurement of ionized calcium levels in colic horses. Moreover correction of hypocalcaemia seems to improve clinical outcome.

Aortic rupture and aorto-pulmonary fistulation in the Friesian horse: Characterisation of the clinical and gross post mortem findings in 24 cases

REASONS FOR PERFORMING STUDY In horses, aortic sinus of Valsalva aneurysms or tears in the aortic root are well-recognised conditions in breeding stallions, often leading to sudden death. A more uncommon form of aortic rupture, located proximal to the ligamentum arteriosum has been reported in 3 Friesian horses. OBJECTIVES The purpose of this study was to phenotypically characterise aortic rupture and aorto-pulmonary fistulation in Friesian horses in terms of clinical and post mortem data based on 24 cases. METHODS Friesian horses that were diagnosed with aortic rupture and aorto-pulmonary fistulation over a period of 13 years (1997-2010) at the Department of Equine Sciences of Utrecht University (n = 15) and Wolvega Equine Hospital (n = 9), were included in this study. Case history, results of clinical examination and gross post mortem findings were screened and analysed. RESULTS Some cases were found dead without prior symptoms, but in several cases signs such as recurrent colic, peripheral oedema and sustained tachycardia were present for several weeks prior to cardiac failure. Clinical examination during hospitalisation revealed increased rectal temperature, peripheral oedema and increased jugular pulse with a bounding arterial pulse. In the majority of horses an aortic rupture of the aortic arch near the ligamentum arteriosum, concurrent with a circumferential cuff of perivascular haemorrhage and aorto-pulmonary fistulation, was found at post mortem examination. CONCLUSIONS Aorto-pulmonary fistulation in conjunction with aortic rupture is more common in Friesians than previously estimated. In some cases findings demonstrate a progressive pathology rather than acute cardiac failure and sudden death. An appropriate approach is necessary during post mortem examination of the heart in order not to overlook the diagnosis. POTENTIAL RELEVANCE Equine practitioners should realise that in Friesian horses presented with a history of recurrent false colic, coughing, sustained tachycardia and/or peripheral oedema, aortic rupture and aorto-pulmonary fistulation should be included in the differential diagnosis.

Thoracic Aortic Rupture and Aortopulmonary Fistulation in the Friesian Horse: Histomorphologic Characterization

Aortic rupture in horses is a rare condition. Although it is relatively common in the Friesian breed, only limited histopathologic information is available. Twenty Friesian horses (1–10 years old) were diagnosed with aortic rupture by postmortem examination. Ruptured aortic walls were analyzed with histology and immunohistochemistry. Based on the histologic and immunohistochemical findings, these cases were divided into 3 groups: acute (n = 4, 20%), subacute (n = 8, 40%), and chronic (n = 8, 40%). Features common to samples from horses in all groups included accumulation of mucoid material; disorganization and fragmentation of the elastic laminae; aortic medial smooth muscle hypertrophy; and medial necrosis of varying degrees, ranging from mild and patchy in the acute cases to severe midzonal necrosis in the chronic cases. Inflammation, most likely secondary to medial necrosis, varied from predominantly neutrophilic infiltrates in the media and periadventitial tissue in the acute group to the presence of mainly hemosiderophages in the periadventitial tissue in the chronic group. Medial fibrosis with aberrant collagen morphology was seen in the subacute group and, more commonly, in the chronic group. Only minimal changes were seen in the aortic vasa vasorum. Smooth muscle hypertrophy and accumulation of mucoid material were not related to the age of the lesions. The findings of this study suggest that a connective tissue disorder affecting elastin or collagen in the aortic media is potentially the underlying cause of aortic rupture in Friesian horses.

Contractile effects of 5‐hydroxytryptamine and 5‐carboxamidotryptamine in the equine jejunum

The use of human prokinetic drugs in colic horses leads to inconsistent results. This might be related to differences in gastrointestinal receptor populations. The motor effects of 5‐hydroxytryptamine (5‐HT; serotonin) on the equine mid‐jejunum were therefore studied. Longitudinal muscle preparations were set up for isotonic measurement. 5‐HT induced tonic contractions with superimposed phasic activity; these responses were not influenced by tetrodotoxin and atropine, suggesting a non‐neurogenic, non‐cholinergic pathway. The 5‐HT receptor antagonists GR 127935 (5‐HT1B,D), ketanserin (5‐HT2A), SB 204741 (5‐HT2B), RS 102221 (5‐HT2C), granisetron (5‐HT3), GR 113808 (5‐HT4) and SB 269970 (5‐HT7) had no influence on the 5‐HT‐induced response; the 5‐HT1A receptor antagonists NAN 190 (pKb=8.13±0.06) and WAY 100635 (pKb=8.69±0.07), and the 5‐HT1,2,5,6,7 receptor antagonist methysergide concentration‐dependently inhibited the 5‐HT‐induced contractile response. The 5‐HT1,7 receptor agonist 5‐carboxamidotryptamine (5‐CT) induced a contractile response similar to that of 5‐HT; its effect was not influenced by tetrodotoxin and atropine, and SB 269970, but antagonised by WAY 100635. 8‐OHDPAT, buspiron and flesinoxan, which are active at rat and human 5‐HT1A receptors, had no contractile influence. These results suggest that the contractile effect of 5‐HT in equine jejunal longitudinal muscle is due to interaction with muscular 5‐HT receptors, which cannot be characterised between the actually known classes of 5‐HT receptors.

Comparative analysis of serotonin in equine plasma with liquid chromatography–tandem mass spectrometry and enzyme-linked immunosorbent assay

Serotonin is regularly measured in equine platelet-poor plasma in research settings. However, reported reference values vary between studies, partially because plasma serotonin concentrations are very low and a reliable and affordable detection method is lacking. A simple, rapid, and sensitive method for serotonin determination in equine platelet-poor plasma using liquid chromatography–tandem mass spectrometry (LC-MS/MS) was developed and validated. Results of a commercially available enzyme-linked immunosorbent assay (ELISA) were compared to the LC-MS/MS results, in order to validate a test more suitable for use in a clinical situation. For LC-MS/MS, 500 µl of plasma was required, and deuterated serotonin was used as an internal standard. The sample preparation was based upon a simple liquid extraction into ethyl acetate. Chromatographic separation was performed with an acetic acid–acetonitrile mobile phase gradient elution. Linearity was demonstrated between 3 ng/ml and 100 ng/ml. A limit of quantification of 3 ng/ml was achieved, corresponding to a limit of detection of 0.10 ng/ml. Comparison of LC-MS/MS and ELISA with Passing–Bablok regression and Bland–Altman plotting showed a poor agreement between the 2 methods, with an increasing difference within the higher range of measurements. Caution is needed when extrapolating results from sources using different analytical techniques.

Determination of the source of increased serotonin (5-HT) concentrations in blood and peritoneal fluid of colic horses with compromised bowel.

REASONS FOR PERFORMING STUDY Increased plasma (5-HT) concentrations are reported in horses predisposed to develop laminitis and after i.v. infusion of endotoxins. In the equine jejunum contractile 5-HT1A-like receptors show tachyphylaxia upon prolonged activation with 5-HT. Therefore, increased systemic 5-HT release in colic horses could play a possible role in the pathophysiology of ileus. OBJECTIVE To investigate possible increased systemic release of 5-HT in colic horses with compromised bowel and to identify the source of 5-HT overload. METHODS Concentrations of 5-HT were determined in plasma and peritoneal fluid (PF) of healthy horses (n = 10), strangulating small intestinal colic horses (n = 18), nonsurgical colic horses (n = 10) and cryptorchid stallions (n = 6). It was attempted to identify the source of 5-HT overload by comparing the blood and PF 5-HT concentrations within horses and by assessing the in vivo platelet activation through determination of the beta-thromboglobulin (beta-TG)/platelet factor 4 (PF4) ratio. RESULTS All horses in the strangulating small intestinal colic group had plasma (P = 0.006) and PF (P = 0.01) 5-HT concentrations above those found in the control group. Plasma beta-TG/PF4 ratio in these horses exceeded 2 in all cases, indicating in vivo platelet activation. Concentrations of 5-HT in PF of colic horses with compromised bowel were significantly lower than the corresponding plasma concentrations (P = 0.005). POTENTIAL RELEVANCE In horses with compromised bowel, significant amounts of 5-HT can be released into the systemic circulation, through massive release of platelet-stored 5-HT. 5-HT is a very potent proinflammatory, vasoconstrictive and immunomodulatory agent. In view of the rapid and prolonged tachyphylaxia, shown for the jejunal 5-HT1A-like receptors, this increased systemic 5-HT release could play a role in the pathophysiology of ileus in horses.

Human skin permeation of emerging mycotoxins (beauvericin and enniatins)

Currently, dermal exposure data of cyclic depsipeptide mycotoxins are completely absent. There is a lack of understanding about the local skin and systemic kinetics and effects, despite their widespread skin contact and intrinsic hazard. Therefore, we provide a quantitative characterisation of their dermal kinetics. The emerging mycotoxins enniatins (ENNs) and beauvericin (BEA) were used as model compounds and their transdermal kinetics were quantitatively evaluated, using intact and damaged human skin in an in vitro Franz diffusion cell set-up and ultra high-performance liquid chromatography (UHPLC)-MS analytics. We demonstrated that all investigated mycotoxins are able to penetrate through the skin. ENN B showed the highest permeation (kp,v=9.44 × 10−6 cm/h), whereas BEA showed the lowest (kp,v=2.35 × 10−6 cm/h) and the other ENNs ranging in between. Combining these values with experimentally determined solubility data, Jmax values ranging from 0.02 to 0.35 μg/(cm2 h) for intact skin and from 0.07 to 1.11 μg/(cm2 h) for damaged skin were obtained. These were used to determine the daily dermal exposure (DDE) in a worst-case scenario. On the other hand, DDE’s for a typical occupational scenario were calculated based on real-life mycotoxin concentrations for the industrial exposure of food-related workers. In the latter case, for contact with intact human skin, DDE’s up to 0.0870 ng/(kg BW × day) for ENN A were calculated, whereas for impaired skin barrier this can even rise up to 0.3209 ng/(kg BW × day) for ENN B1. This knowledge is needed for the risk assessment after skin exposure of contaminated food, feed, indoor surfaces and airborne particles with mycotoxins.

Morphological changes in the small intestinal smooth muscle layers of horses suffering from small intestinal strangulation: is there a basis for predisposition for reduced contractility?

REASONS FOR PERFORMING STUDY Intestinal strangulation often leads to enterectomy after which ileus can develop. This has prompted research to look into possible pathophysiological processes triggering equine ileus. However, morphological changes of the small intestinal smooth muscle in relation to equine colic have not yet been studied. OBJECTIVES The presence of some smooth muscle proteins was morphologically assessed and quantified in control and colic horses. In addition, the up- or down-regulation of heat shock proteins (HSP20 and HSP27) influencing the contractility of smooth muscles was studied. METHODS Cranial resection margins of 18 strangulated small intestinal samples were collected. Small intestinal control samples were collected from 11 horses subjected to euthanasia for other than gastrointestinal-related reasons. Formaldehyde-fixed tissue was paraffin-embedded and processed for conventional staining and immunohistochemistry. Snap-frozen full-thickness biopsies were collected for western blot analyses. RESULTS Evaluating the muscle layer microscopically, colic samples showed significantly more signs of degradation than controls (P = 0.026) of which vacuolar degeneration was most prominent (P = 0.009). In colic samples, myosin protein levels were decreased (P = 0.022) whereas desmin (P = 0.049) and HSP20 protein levels (P = 0.005) were elevated. CONCLUSIONS In colic samples, microscopic lesions at the level of the muscle layer indicate a stress response. In addition, modified amounts of structural proteins such as myosin and desmin together with increased HSP20 levels could perhaps provide a basis for explaining the malfunctioning of the intestinal muscle layer. POTENTIAL RELEVANCE Post operative ileus, following small intestinal strangulation and resection, could be related in part to a dysfunctional muscle layer. In addition to microscopic signs of degeneration, myosin and HSP20 were affected. Pharmacological interventions might alter HSP20 expressions and thus serve a protective effect.

Biomechanical and biochemical properties of the thoracic aorta in warmblood horses, Friesian horses, and Friesians with aortic rupture.

BackgroundThoracic aortic rupture and aortopulmonary fistulation are rare conditions in horses. It mainly affects Friesian horses. Intrinsic differences in biomechanical properties of the aortic wall might predispose this breed. The biomechanical and biochemical properties of the thoracic aorta were characterized in warmblood horses, unaffected Friesian horses and Friesians with aortic rupture in an attempt to unravel the underlying pathogenesis of aortic rupture in Friesian horses. Samples of the thoracic aorta at the ligamentum arteriosum (LA), mid thoracic aorta (T1) and distal thoracic aorta (T2) were obtained from Friesian horses with aortic rupture (A), nonaffected Friesian (NA) and warmblood horses (WB). The biomechanical properties of these samples were determined using uniaxial tensile and rupture assays. The percentages of collagen and elastin (mg/mg dry weight) were quantified.ResultsData revealed no significant biomechanical nor biochemical differences among the different groups of horses. The distal thoracic aorta displayed an increased stiffness associated with a higher collagen percentage in this area and a higher load-bearing capacity compared to the more proximal segments.ConclusionsOur findings match reported findings in other animal species. Study results did not provide evidence that the predisposition of the Friesian horse breed for aortic rupture can be attributed to altered biomechanical properties of the aortic wall.