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Endothelin 1 in healthy foals and in foals affected by neonatal diseases
Theriogenology 84 (2015) 667–673
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Theriogenology journal homepage: www.theriojournal.com
Endothelin 1 in healthy foals and in foals affected by neonatal diseases A. Giordano a, b, C. Castagnetti c, S. Panzani d, *, S. Paltrinieri a, b, F. Freccero c, M.C. Veronesi d a
Department of Veterinary Science and Public Health, University of Milan, Milan, Italy Veterinary Teaching Hospital, Polo Veterinario di Lodi, University of Milan, Lodi, Italy Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell’Emilia, Bologna, Italy d Department of Health, Animal Science and Food Safety, University of Milan, Milan, Italy b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2015 Received in revised form 27 April 2015 Accepted 28 April 2015
In newborn babies, endothelin 1 (ET-1), a potent vasoconstrictor, increases during septicemia and severe respiratory syndromes. Because equine neonatal sepsis (ENS) and perinatal asphyxia syndrome (PAS) are major causes of morbidity and mortality in newborn foals and because no information on the concentration of ET-1 in healthy and sick foals has been reported yet, the aims of this study were (1) to define the serum concentration of Big ET-1 in healthy neonatal foals during the first week of age; (2) to preliminarily explore the diagnostic and prognostic role of Big ET-1 during ENS and PAS. Six healthy and 23 sick foals affected by ENS and/or PAS were enrolled in the study. In healthy foals, Big ET-1 concentration increased in the first hours of life until 24 hours after birth, and it remained constant during the first 3 days, then gradually decreased becoming significantly lower from Day 4 onward (P < 0.05). In sick foals, only 26.1% of animals showed higher values of Big ET-1 than controls at admission, and no difference between surviving and nonsurviving foals was found. Because in nonsurviving foals, Big ET-1 remained over the maximum value recorded in clinically healthy horses or, when normal at admission, increased over time; this study suggested that repeated measurement of Big ET-1 during hospitalization may be helpful in monitoring the course of the disease. In conclusion, possible prognostic information may be obtained by repeated analysis of Big ET-1 during hospitalization, but further studies are needed. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Endothelin 1 Equine neonatal sepsis Perinatal asphyxia syndrome Newborn foal
1. Introduction Endothelin 1 (ET-1) is a potent vasoconstrictor synthesized mainly by endothelial cells in response to several stimuli that include proinflammatory cytokines, hormones, angiotensin II, hypoxia, and vasodilation [1]. Endothelin 1 contributes to maintain the baseline vascular tone or to increase systemic pressure [1,2] by stimulating specific receptors expressed on smooth muscle cells or on
* Corresponding author. Tel.: þ39 02 50318149; fax: þ39 02 50318148. E-mail address: [email protected] (S. Panzani). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.04.026
myocardiocytes. Moreover, ET-1 modulates the interaction between endothelial cells and leukocytes [3] and the production of cytokines [4]. In turn, sepsis and endotoxemia may damage endothelial cells and induce overproduction of ET-1 [5]. Endothelin 1 is considered a useful marker in human neonatology because its serum concentration in newborns is particularly high compared to adults, increases in newborns with sepsis, and correlates with the severity of the disease [6]. Additionally, high serum concentrations of ET-1 have been reported in asphyctic newborns [7] or in newborns with persistent pulmonary hypertension [8]. Endothelin 1 can be measured in domestic animals using reagents designed for human ET-1 because of a high
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interspecies homology of ET-1 [9]. Measurement of the precursor Big ET-1 that has a longer half-life and well correlates with the concentration of ET-1 is usually preferred [10]. Big ET-1 has been measured in cardiopathic dogs and cats [10,11], in dogs with chronic kidney disease [12] or endotoxic shock [13]. In horses, ET-1 has been studied during laminitis [14,15], joint diseases [16], colic syndromes [17], and respiratory disorders [18–20]. Equine neonatal sepsis (ENS) and perinatal asphyxia syndrome (PAS) are the major causes of morbidity and mortality in newborn foals [21]. Equine neonatal sepsis occurs in the first 2 weeks of life and is characterized by the release of proinflammatory cytokines that induce a systemic inflammatory reaction syndrome, which in turn causes endothelial damage and release of vasoactive mediators [22] and may progress to septic shock and death. Vasoactive mediators induce pulmonary hypertension, followed by a hypotensive status typical of the endotoxic shock [23]. Perinatal asphyxia syndrome may depend on maternal, placental, or fetal [24] causes. Hypoxia affects primarily the central nervous system and induces a variety of clinical signs that depend on the type and duration of hypoxia. Moreover, tissue hypoxia may induce systemic inflammatory reaction syndrome [25]. Both conditions (ENS and PAS) can, therefore, lead to a possible overproduction of ET-1. The early diagnosis of ENS and PAS is essential for a successful outcome; thus, the availability of biomarkers that support a clinical diagnosis or provide prognostic information may be useful in the management of sick foals. Because the authors are not aware of other studies on the concentration of ET-1 in clinically healthy foals or in foals with ENS or PAS, the aims of this study were to define the serum concentration of Big ET-1 in healthy neonatal foals of different ages and to explore the potential of Big ET-1 as a diagnostic and prognostic biomarker of neonatal septicemia and asphyxia. 2. Materials and methods The study has been conducted during the breeding seasons 2013. All procedures on the animals were carried out with the approval of the Ethical Committee of the University of Bologna, in accordance with DL 116/92, approved by the Ministry of Health. The owners gave oral informed consent. 2.1. Control group Six light horse foals (Equus caballus), two females, and four males, born by spontaneous delivery at the Equine Perinatology Unit of the Department of Veterinary Medical Sciences of the University of Bologna, were included in the study. All foals should fulfill some requirements: they should be full term, with normal size, coat, and fetlock joint extension. The mean Apgar index within 10 minutes of birth should be greater than eight, and all foals must show normal physical and behavioral characteristics, as previously described to assess maturity and viability [26,27].
No clinical signs or laboratory changes consistent with ENS, PAS, or with any other disease should be found during the period of observation. For the study purposes, blood was collected from of each foal via the jugular vein, with the following schedule: 3 hours from birth (T0); 12 hours from birth (T1); 24 hours from birth (T2); 2 days (T3); 3 days (T4); 4 days (T5); 5 days (T6); 6 days (T7); 7 days (T8). Blood was collected in plain tubes and, immediately after clotting, serum was separated by centrifugation (2500 g for 10 minutes) and stored at 20 C until analyses. 2.2. Sick foals Twenty-three light breed sick foals, aged less than 4 days old (mean age: 28.4 25 hours), admitted to the Equine Perinatology Unit of the Department of Veterinary Medical Sciences of the University of Bologna were included in the sick group. The inclusion criterion for the pathologic group was the diagnosis of ENS and/or PAS requiring level 2 or 3 of intensive care [27]. Level 2 care is provided to neonates that are quite severely affected; they may be unable to stand or to nurse from the mare and need round-the-clock care. Usually this level of care involves separation of the foal from the dam. Level 3 care is specific for extremely compromised foals that usually have multisystem dysfunction; they need round-the-clock care and must be assisted by specialists. Foals were classified as affected by ENS when showed a positive blood culture and/or a sepsis score greater than 11 [28], whereas PAS was diagnosed on the basis of history and clinical signs, such as the presence of neurologic dysfunction without other neurologic diseases or trauma [29]. Blood culture was performed regardless of antibiotic therapy before referral using 10 mL of jugular blood withdrawn after clipping and aseptic preparation of the skin. The sampling needle was then discarded, and a new needle was used to inoculate the blood into the commercially available culture bottle (Oxoid Signal Blood Culture System; Oxoid Limited). Ten foals were classified as affected by ENS, eight by PAS, whereas five foals suffered both the diseases. From admission until discharge or death, animals were constantly monitored by clinical, laboratory, and instrumental examinations, according to the severity of the disease. Each foal received proper treatments in relation to its specific clinical presentation; any resuscitation treatment (that could influence ET-1 concentrations) was recorded. Sick foals were sampled every 24 hours from the day of admission until discharge (n ¼ 12) or death (n ¼ 8, four with ENS, three with ENS and PAS, and one with PAS). The first sample was always collected before any medical treatment. Only 20 foals were repeatedly sampled during hospitalization, since three foals died soon after admission. Blood was collected in plain tubes and, immediately after clotting, serum was separated by centrifugation
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Fig. 1. Values of Big endothelin 1 recorded in six healthy foals during the first week of life. Boxes indicate the I to II interquartile interval, the horizontal line corresponds to the median, the vertical lines are the limits of outlier distribution according to the Tukey rule. Near outliers are indicated by the symbol “þ”. The same letter indicates values not statistically different; different letters correspond to P < 0.05.
(2500 g for 10 minutes) and stored at 20 C until analyses. 2.3. Endothelin 1 measurement Big ET-1 was measured in all the samples using a sandwich ELISA (IBL Hamburg). The kit includes a primary capturing antibody against the C-terminal 22 to 38 amino acid sequence of rat Big ET-1 which shows closer homology to the equine amino acid sequence [9] and a secondary antibody for detection, directed against a different antigenic site of the molecule. As a standard material to generate a calibration curve, human big ET-1 is included in the kit and the tetramethylbenzidine as a chromogen. The procedure was performed following the recommendation of the manufacturer of the kit, and the plates were read using an automated plate reader (Dasit multiscan, Dasit) at a wavelength of 450 nm. Intra-assay and interassay coefficients of variation reported by the manufacturer are lower than 5% and 13%, respectively at each value. However, to reduce the interassay variability, all samples were processed in the same batch. 2.4. Statistical analysis Statistical analysis was performed using Analyse-it software for Microsoft Excel. P values less than 0.05 were considered to be statistically significant. Results recorded in healthy foals at different sampling times were compared using a nonparametric ANOVA test for paired data (Friedman test) followed by the Wilcoxon signed-rank test to assess the difference between each time point. The
frequency of abnormal values (i.e., values higher than the maximum value recorded in healthy foals in the first 3 days of life) recorded at admission in all sick foals and in foals grouped on the basis of diagnosis was calculated. Finally, to achieve preliminary information about the prognostic value of ET-1, results recorded in nonsurviving foals at admission were compared with those of survivors using a nonparametric Student’s t test for unpaired samples (Mann Whitney U test). 3. Results All six healthy foals fulfilled the required characteristics, and in detail: birth weight ranging between 40 and 58 kg, mean Apgar index within 10 minutes of birth was 9 0.89, the time to stand up was 55.9 10.27 minutes and to the first suckle was 101 13.37 minutes. Results collected from healthy foals in the first 7 days of life are reported in Figure 1. As shown in the Figure 1, the serum concentration of Big ET-1 moderately increases in the first hours of life (17.4 4.0 pg/mL at 3 hours, 22.9 5.5 pg/mL at 12 hours) until 24 hours (20.4 6.4 pg/mL) after birth and then gradually decreases until Days 5, 6, and 7 (mean values around 12 pg/mL), when values are significantly lower than those recorded on Days 1, 2, and 3 (mean values around 18 pg/mL). No significant differences were recorded in the first 3 days of life. On the basis of lack of significant differences in healthy animals in the first five samples, values collected from sick foals at admission (before any medical treatment) were compared with the maximum value recorded in healthy
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foals from T0 to T4 (35.2 pg/mL). However, considering the low number of animals involved, only a descriptive analysis was performed. Such a comparison (Fig. 2) revealed that only 6 of 23 sick foals (26.1%) had values higher than this threshold. This group of foals with high Big ET-1 included 3 of 10 foals with ENS (30%), 2 of 8 foals with PAS (25%), and 1 of 5 foals with ENS and PAS (20%). Six of the sick foals received resuscitation treatment (four foals were treated with dobutamine and two foals with dobutamine and norepinephrine): two with PAS, one with ENS, and three with ENS and PAS. Eight sick foals (four with ENS, three with ENS and PAS, one with PAS) died during hospitalization, five of which received the previously mentioned treatments. Among these, only four (two with ENS, one with PAS, and one with both the diseases) had higher ET-1 values than controls at admission. Nevertheless, the results of nonsurviving foals (mean standard deviation: 23.5 5.7 pg/mL; median: 21.8 pg/mL; I to III interquartile range: 19.4–28.6 pg/mL) were not significantly different (P ¼ 0.123) from those of survivors (29.9 25.2 pg/mL, 18.6 pg/mL, 13.3–54 pg/mL, respectively). The analysis of sequential results (Fig. 3) showed that only one of eight nonsurviving foal had Big ET-1 values persistently below the maximum values recorded in healthy foals. Conversely, the serum concentrations of Big ET-1 remained persistently high or had only transient decreases in the four foals that at admission had abnormal values and increased during hospitalization in the three foals that had normal levels at admission. In surviving foals that had normal values of Big ET-1 at admission, instead, serum levels of Big ET-1 remained within the normal values during the whole hospitalization, whereas in the only one foal with high values at admission that was repeatedly sampled over time, Big ET-1 values rapidly normalized. 4. Discussion
Fig. 2. Distribution of values recorded in 23 sick foals at admission. The dotted gray line indicates the upper value recorded in clinically healthy foals aged less than 4 days. Big-ET-1, Big endothelin 1.
Despite the frequent occurrence of ENS and PAS in newborn foals, studies about prognostic markers of both these conditions are scarce. In human medicine, sepsis and asphyxia are known to induce increases in the serum concentration of ET-1 either in adults or in newborns, where increases of ET-1 or of its precursor Big ET-1 may provide prognostic information [6,7,30,31]. Conversely, no information is reported on ET-1 in foals affected by ENS and/or PAS and, more importantly, no information is available on the possible age-related changes of the serum concentration of this biomarker in healthy neonatal foals, so this represents the first study on the possible use of ET-1 for health status evaluation in newborn foals. Therefore, the first step of the study was focused on investigating the possible presence of changes in the serum concentration of Big ET-1 in the first days of life in healthy foals. Because, unfortunately, this part of the study was biased by the low number of animals, it was not possible to define appropriate reference intervals for each sampling time, using the approaches recommended by the current guidelines [32,33]. However, the statistical
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Fig. 3. Values recorded during hospitalization in sick foals. Foals with septicemia are indicated with the squares; foals with asphyxia are indicated with circles; foals with septicemia and asphyxia are indicated with the triangles. Black symbols indicate nonsurviving foals; white symbols indicate surviving foals. The dotted gray line indicates the upper value recorded in healthy foals less than 4 days old. Big ET-1, Big endothelin 1.
comparison of the results obtained in healthy foals in the first week of life revealed a trend that parallels what is reported in human newborns, where Big ET-1 values increase in the first hours of life and then start to decrease after 5 days until Day 30 [34,35]. A similar trend has been also reported in piglets [36]. In our caseload, even on Day 7, values were still higher than those reported in adult horses in studies that, however, used a different method for measuring Big ET-1 [17,18]. Because ET-1 is elevated in fetuses, where it contributes to maintain an adequate pulmonary vasodilation at birth followed by a progressive reduction of vascular resistance after birth [37], this agerelated change may depend on an incomplete removal of circulating ET-1 by the liver of newborns, less functional than in adults [31]. As already reported for other molecules involved in inflammation or tissue damages [38,39], independently from the mechanism responsible for these changes, the finding of higher serum concentration of Big ET-1 in newborn foals than in adults evidences the need to establish, in the future, age-specific reference intervals. This would avoid to erroneously interpreting as extremely high Big ET-1 values that actually depend on the young age of the foals. Results from sick foals, however, evidenced that the diagnostic or prognostic role of increased Big ET-1 levels at admission (before any medical treatment) seems to be moderate, because approximately one fourth (26.1%) of sick foals had values higher than the maximum value recorded in clinically healthy foals. Moreover, increases of serum Big ET-1 seem not to be associated with a specific condition because the group of foals with abnormal values included foals with ENS, PAS, or both. Additionally, although the majority of foals with abnormal levels of Big ET-1 (four of six) died during hospitalization, 4 of 8 nonsurviving foals had normal values at admission and no significant differences were found
between survivors and nonsurvivors. All these findings seem to contrast with what reported in human medicine, where high ET-1 values correlate with a poor prognosis either in sepsis or in conditions characterized by hypoxia [30,31,40]. Also, previous studies on adult horses reported that ET-1 may be a marker of severity of inflammatory conditions and negatively correlated with survival [17]. Therefore, it may be postulated that the magnitude of ET-1 responses to endotoxic shock or hypoxia in foals is less intense than in adult horses or in other species. Alternatively, the lack of differences between sick and healthy foals or between surviving and nonsurviving foals may depend on the short time elapsed between the onset of clinical signs and the first sampling or on different degree of severity of tissue damages in affected horses. Figueras-Aloy et al. [6] reported that the highest ET-1 levels in neonatal sepsis appeared at 3 to 5 days after the diagnosis and later decreased. Only newborns with severe sepsis presented a significant increase in ET-1 concentrations from the beginning of the septicemic process. However, any conclusion about the possible prognostic value of a single measurement of serum ET-1 at admission should be reviewed when age-specific reference intervals are established on a larger cohort of healthy neonatal foals. Conversely, this study reported that repeated measurement of Big ET-1 during hospitalization may be helpful in monitoring the course of the disease: Because of the heterogeneity of samplings collected during hospitalization (the different foals were admitted at different ages, foals received different therapies, such as resuscitation treatments, specific for their clinical conditions, and the length of hospitalization was variable), results recorded over time were not statistically compared to each other. However, with a single exception, Big ET-1 values in nonsurvivors, independently on the disease (PAS and/or ENS), remained over the maximum value recorded in healthy
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foals or, when normal at admission, increased over time. Five of the six animals that received resuscitation therapies did not survive; therefore, treatment was ineffective and may have contributed to the increase of Big ET-1 during hospitalization because it is known that in humans, during septic shock, the release or the production of endothelin may increase as a consequence of the infusion of catecholamines [41]. It must be stressed that the significance of high values in sequential samplings is stronger than the single value at admission. In healthy foals, in fact, the serum concentration of Big ET-1 decreases with age, and therefore, the difference between values recorded in healthy and in sick foals become even more evident as age increases. These results indicate that, when the disease progresses despite the administration of any kind of treatment, stimuli inducing the release of ET-1 from damaged endothelium persist and maintain the serum concentration of Big-ET-1 elevated.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
4.1. Conclusions [12]
In conclusion, this study evidenced that serum concentration of Big ET-1 in foals shares some similarity with that of human ET-1. In particular, age-related changes found in the first days of life are the same as recorded in human newborns. The low number of animals involved in the study is the most important limitation of the work to arrive to strong conclusions. However, considering that this is the first reference of using ET-1 for health status evaluation in foals and the promising results obtained, this work underlines the need of further investigations to create specific age-related reference ranges for the equine newborn. This information will be relevant on a diagnostic perspective, to avoid erroneously interpreting as high values that are actually normal in newborns. Moreover, because in nonsurviving foals, Big ET-1 remained over the maximum value recorded in clinically healthy horses or, when normal at admission, increased over time, this study suggested that repeated measurement of Big ET-1 during hospitalization may be helpful in monitoring the course of the disease. Prognostic information may be obtained by repeated analysis of Big ET-1 during hospitalization, but further studies are needed.
[13]
[14] [15] [16]
[17]
[18]
[19]
[20]
[21]
[22]
Competing Interests [23]
None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the article.
[24] [25]
References [1] Rubanyi GM, Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 1994;46:325–415. [2] Haynes WG. Endothelins as regulators of vascular tone in man. Clin Sci 1995;88:509–17. [3] Murohara T, Lefer AM. Autocrine effects of endothelin-1 on leukocyte-endothelial interaction: stimulation of endothelin B receptor subtype reduces endothelial adhesiveness via a nitric oxide dependent mechanism. Blood 1996;88:3894–900. [4] Finsnes F, Lyberg T, Christensen G, Skjønsberg OH. Effect of endothelin antagonism on the production of cytokines in eosinophilic
[26]
[27]
[28]
[29]
airway inflammation. Am J Physiol Lung Cell Mol Physiol 2001;280: L659–665. Ros J, Leivas A, Jiménez W, Morales M, Bosch-Marcé M, Arroyo V, et al. Effect of bacterial lipopolysaccharide on endothelin-1 production in human vascular endothelial cells. J Hepatol 1997; 26:81–7. Figueras-Aloy J, Gómez-Lopez L, Rodríguéz-Miguélez JM, JordánGarcía Y, Salvia-Roiges MD, Jiménez W, et al. Plasma endothelin-1 and clinical manifestations of neonatal sepsis. J Perinat Med 2004; 32:522–6. Kojima T, Isozaki-Fukuda Y, Takedatsu M, Ono A, Hirata Y, Kobayashi Y. Plasma endothelin-1 like immunoreactivity levels in neonates. Eur J Pediatr 1992;151:913–5. Abman SH. Recent advances in the pathogenesis and treatment of persistent pulmonary hypertension of the newborn. Neonatology 2007;91:283–90. Uchide T, Fujimori Y, Temma K, Sasaki T, Kizaki K, Hara Y, et al. cDNA cloning, sequence analysis and organ distribution of horse preproendothelin-2. J Cardiovasc Pharmacol 2004;44:S430–4. O’Sullivan ML, O’Grady MR, Minors SL. Plasma big endothelin-1, atrial natriuretic peptide, aldosterone, and norepinephrine concentrations in normal Doberman pinschers and Doberman pinschers with dilated cardiomyopathy. J Vet Int Med 2007;21:92–9. Prosek R, Sisson DD, Oyama MA, Biondo AW, Solter PE. Measurements of plasma endothelin immunoreactivity in healthy cats and cats with cardiomyopathy. J Vet Int Med 2004;18:826–30. Rossi G, Giordano A, Breda S, Lisi C, Roura X, Zatelli A, et al. Bigendothelin 1 (Big ET-1) and homocysteine in the serum of dogs with chronic kidney disease. Vet J 2013;198:109–15. Nakamura T, Kasai K, Sekiguchi Y, Banba N, Takahashi K, Hattori Y, et al. Elevation of plasma endothelin concentrations during endotoxin shock in dogs. Eur J Pharmacol 1991;205:277–82. Hood DM, Amoss MS, Grosenbaugh DA. Equine laminitis: a potential model of Raynaud’s phenomenon. Angiology 1990;41:270–7. Katwa LC, Johnson PJ, Ganjamt VK, Kreeger JM, Messer NT. Expression of endothelin in equine laminitis. Equine Vet J 1999;31:243–7. LeBlanc C, Moore RM. Plasma and synovial fluid endothelin-1 and nitric oxide concentrations in horses with and without joint disease. Am J Vet Res 2002;63:1648–54. Ramaswamy CM, Eades SC, Vegopa l CS, Hosgood GL, Garza F, Barker SA, et al. Plasma concentrations of endothelin-like immunoreactivity in healthy horses and horses with naturally acquired gastrointestinal tract disorders. Am J Vet Res 2002;63:454–9. Benamou AE, Art T, Marlin DJ, Roberts CA, Lekeux P. Variations in systemic and pulmonary endothelin-1 in horses with recurrent airway obstruction (heaves). Pulm Pharmacol Ther 1998; 11:231–5. Benamou AE, Marlin DJ, Lekeux P. Endothelin in the equine hypoxic pulmonary vasoconstrictive response to acute hypoxia. Equine Vet J 2001;33:345–53. Venugopal CS, Polikepahad S, Holmes EP, Heuvel JV, Leas TL, Moore RM. Endothelin receptor alterations in equine air way hyperreactivity. Can J Vet Res 2006;70:50–7. Galvin NP, Corley KTT. Causes of disease and death from birth to 12 months of age in the Thoroughbred horse in Ireland. Irish Vet J 2010;63:37–43. Wort SJ, Evans TW. The role of the endothelium in modulating vascular control in sepsis and related conditions. Br Med Bull 1999; 55:30–48. Lavoie JP, Madigan JE, Cullor JS, Powell WE. Haemodynamic, pathological, haematological and behavioural changes during endotoxin infusion in equine neonates. Equine Vet J 1990;22:23–9. Galvin N, Collins D. Perinatal asphyxia syndrome in the foal: review and a case report. Irish Vet J 2004;57:707–14. Wong D, Wilkins PA, Bain FT, Brockus C. Neonatal encephalopathy in foals. Compend Contin Educ Vet 2011;33:1–10. Rossdale PD, Ousey JC, Silver M, Fowden A. Studies on equine prematurity 6: guidelines for assessment of foal maturity. Equine Vet J 1984;16:300–2. Koterba AM. Physical examination. In: Koterba AM, Drummond WH, Kosch PC, editors. Equine clinical neonatology. Philadelphia, PA: Lea & Fabiger; 1990. p. 71–86. Brewer BD, Koterba AM. Development of a scoring system for the early diagnosis of equine neonatal sepsis. Equine Vet J 1988;20: 18–22. Knottenbelt DC, Holdstock N, Madigan JE, Knottenbelt DC, Holdstock N, Madigan JE. Neonatal syndromes. In: Equine neonatology: medicine and surgery. St. Louis, MO: Saunders Elsevier; 2004. p. 155–364.
A. Giordano et al. / Theriogenology 84 (2015) 667–673 [30] Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, et al. Definitions for sepsis and organ failure and guidelines for use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 1992;101:1644–55. [31] Paulus P, Jennewein C, Zacharowski K. Biomarkers of endothelial dysfunction: can they help us deciphering systemic inflammation and sepsis? Biomarkers 2011;16:S11–21. [32] Geffré A, Friedrichs K, Harr K, Concordet D, Trumel C, Braun JP. Reference values: a review. Vet Clin Pathol 2009;38:288–98. [33] Friedrichs KR, Harr KE, Freeman KP, Szladovits B, Walton RM, Barnhart KF, et al. American Society for Veterinary Clinical Pathology. ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics. Vet Clin Pathol 2012;41:441–53. [34] Nisell H, Hemsén A, Lunell NO, Wolff K, Lundberg MJ. Maternal and fetal levels of a novel polypeptide, endothelin: evidence for release during pregnancy and delivery. Gynecol Obstet Invest 1990;30:129–32. [35] Endo A, Shimada M, Ayusawa M, Minato M, Takada M, Takahashi S, et al. Nitric oxide and endothelin-1 during postnatal life. Biol Neonate 1996;70:15–20.
673
[36] Levy M, Tulloh RM, Komai H, Stuart-Smith K, Haworth SG. Maturation of the contractile response and its endothelial modulation in newborn porcine intrapulmonary arteries. Pediatr Res 1995;38:25–9. [37] Gao Y, Raj U. Regulation of the pulmonary circulation in the fetus and newborn. Physiol Rev 2010;90:1291–335. [38] Paltrinieri S, Giordano A, Villani M, Manfrin M, Panzani S, Veronesi MC. Influence of age and type of foaling plasma protein electrophoresis (PPE) and serum amyloid A (SAA) concentration and preliminary results on the role of PPE and SAA as markers of equine neonatal septicaemia. Vet J 2008;176:393–6. [39] Castagnetti C, Pirrone A, Mariella J, Mari G. Venous blood lactate evaluation in equine neonatal intensive care. Theriogenology 2010; 73:343–57. [40] Bennet J, Cooper D, Balakrishnan A, Rhodes M, Lewis M. Is the role for serum endothelin in predicting the severity of acute pancreatitis? Hepatobiliary Pancreat Dis Int 2006;5:290–3. [41] Voerman HJ, Stehouwer CD, van Kamp GJ, Strack van Schijndel RJ, Groeneveld AB, Thijs LG. Plasma endothelin levels are increased during septic shock. Crit Care Med 1992;20: 1097–101.
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Endothelin 1 in healthy foals and in foals affected by neonatal diseases A. Giordano a, b, C. Castagnetti c, S. Panzani d, *, S. Paltrinieri a, b, F. Freccero c, M.C. Veronesi d a
Department of Veterinary Science and Public Health, University of Milan, Milan, Italy Veterinary Teaching Hospital, Polo Veterinario di Lodi, University of Milan, Lodi, Italy Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell’Emilia, Bologna, Italy d Department of Health, Animal Science and Food Safety, University of Milan, Milan, Italy b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2015 Received in revised form 27 April 2015 Accepted 28 April 2015
In newborn babies, endothelin 1 (ET-1), a potent vasoconstrictor, increases during septicemia and severe respiratory syndromes. Because equine neonatal sepsis (ENS) and perinatal asphyxia syndrome (PAS) are major causes of morbidity and mortality in newborn foals and because no information on the concentration of ET-1 in healthy and sick foals has been reported yet, the aims of this study were (1) to define the serum concentration of Big ET-1 in healthy neonatal foals during the first week of age; (2) to preliminarily explore the diagnostic and prognostic role of Big ET-1 during ENS and PAS. Six healthy and 23 sick foals affected by ENS and/or PAS were enrolled in the study. In healthy foals, Big ET-1 concentration increased in the first hours of life until 24 hours after birth, and it remained constant during the first 3 days, then gradually decreased becoming significantly lower from Day 4 onward (P < 0.05). In sick foals, only 26.1% of animals showed higher values of Big ET-1 than controls at admission, and no difference between surviving and nonsurviving foals was found. Because in nonsurviving foals, Big ET-1 remained over the maximum value recorded in clinically healthy horses or, when normal at admission, increased over time; this study suggested that repeated measurement of Big ET-1 during hospitalization may be helpful in monitoring the course of the disease. In conclusion, possible prognostic information may be obtained by repeated analysis of Big ET-1 during hospitalization, but further studies are needed. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Endothelin 1 Equine neonatal sepsis Perinatal asphyxia syndrome Newborn foal
1. Introduction Endothelin 1 (ET-1) is a potent vasoconstrictor synthesized mainly by endothelial cells in response to several stimuli that include proinflammatory cytokines, hormones, angiotensin II, hypoxia, and vasodilation [1]. Endothelin 1 contributes to maintain the baseline vascular tone or to increase systemic pressure [1,2] by stimulating specific receptors expressed on smooth muscle cells or on
* Corresponding author. Tel.: þ39 02 50318149; fax: þ39 02 50318148. E-mail address: [email protected] (S. Panzani). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.04.026
myocardiocytes. Moreover, ET-1 modulates the interaction between endothelial cells and leukocytes [3] and the production of cytokines [4]. In turn, sepsis and endotoxemia may damage endothelial cells and induce overproduction of ET-1 [5]. Endothelin 1 is considered a useful marker in human neonatology because its serum concentration in newborns is particularly high compared to adults, increases in newborns with sepsis, and correlates with the severity of the disease [6]. Additionally, high serum concentrations of ET-1 have been reported in asphyctic newborns [7] or in newborns with persistent pulmonary hypertension [8]. Endothelin 1 can be measured in domestic animals using reagents designed for human ET-1 because of a high
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interspecies homology of ET-1 [9]. Measurement of the precursor Big ET-1 that has a longer half-life and well correlates with the concentration of ET-1 is usually preferred [10]. Big ET-1 has been measured in cardiopathic dogs and cats [10,11], in dogs with chronic kidney disease [12] or endotoxic shock [13]. In horses, ET-1 has been studied during laminitis [14,15], joint diseases [16], colic syndromes [17], and respiratory disorders [18–20]. Equine neonatal sepsis (ENS) and perinatal asphyxia syndrome (PAS) are the major causes of morbidity and mortality in newborn foals [21]. Equine neonatal sepsis occurs in the first 2 weeks of life and is characterized by the release of proinflammatory cytokines that induce a systemic inflammatory reaction syndrome, which in turn causes endothelial damage and release of vasoactive mediators [22] and may progress to septic shock and death. Vasoactive mediators induce pulmonary hypertension, followed by a hypotensive status typical of the endotoxic shock [23]. Perinatal asphyxia syndrome may depend on maternal, placental, or fetal [24] causes. Hypoxia affects primarily the central nervous system and induces a variety of clinical signs that depend on the type and duration of hypoxia. Moreover, tissue hypoxia may induce systemic inflammatory reaction syndrome [25]. Both conditions (ENS and PAS) can, therefore, lead to a possible overproduction of ET-1. The early diagnosis of ENS and PAS is essential for a successful outcome; thus, the availability of biomarkers that support a clinical diagnosis or provide prognostic information may be useful in the management of sick foals. Because the authors are not aware of other studies on the concentration of ET-1 in clinically healthy foals or in foals with ENS or PAS, the aims of this study were to define the serum concentration of Big ET-1 in healthy neonatal foals of different ages and to explore the potential of Big ET-1 as a diagnostic and prognostic biomarker of neonatal septicemia and asphyxia. 2. Materials and methods The study has been conducted during the breeding seasons 2013. All procedures on the animals were carried out with the approval of the Ethical Committee of the University of Bologna, in accordance with DL 116/92, approved by the Ministry of Health. The owners gave oral informed consent. 2.1. Control group Six light horse foals (Equus caballus), two females, and four males, born by spontaneous delivery at the Equine Perinatology Unit of the Department of Veterinary Medical Sciences of the University of Bologna, were included in the study. All foals should fulfill some requirements: they should be full term, with normal size, coat, and fetlock joint extension. The mean Apgar index within 10 minutes of birth should be greater than eight, and all foals must show normal physical and behavioral characteristics, as previously described to assess maturity and viability [26,27].
No clinical signs or laboratory changes consistent with ENS, PAS, or with any other disease should be found during the period of observation. For the study purposes, blood was collected from of each foal via the jugular vein, with the following schedule: 3 hours from birth (T0); 12 hours from birth (T1); 24 hours from birth (T2); 2 days (T3); 3 days (T4); 4 days (T5); 5 days (T6); 6 days (T7); 7 days (T8). Blood was collected in plain tubes and, immediately after clotting, serum was separated by centrifugation (2500 g for 10 minutes) and stored at 20 C until analyses. 2.2. Sick foals Twenty-three light breed sick foals, aged less than 4 days old (mean age: 28.4 25 hours), admitted to the Equine Perinatology Unit of the Department of Veterinary Medical Sciences of the University of Bologna were included in the sick group. The inclusion criterion for the pathologic group was the diagnosis of ENS and/or PAS requiring level 2 or 3 of intensive care [27]. Level 2 care is provided to neonates that are quite severely affected; they may be unable to stand or to nurse from the mare and need round-the-clock care. Usually this level of care involves separation of the foal from the dam. Level 3 care is specific for extremely compromised foals that usually have multisystem dysfunction; they need round-the-clock care and must be assisted by specialists. Foals were classified as affected by ENS when showed a positive blood culture and/or a sepsis score greater than 11 [28], whereas PAS was diagnosed on the basis of history and clinical signs, such as the presence of neurologic dysfunction without other neurologic diseases or trauma [29]. Blood culture was performed regardless of antibiotic therapy before referral using 10 mL of jugular blood withdrawn after clipping and aseptic preparation of the skin. The sampling needle was then discarded, and a new needle was used to inoculate the blood into the commercially available culture bottle (Oxoid Signal Blood Culture System; Oxoid Limited). Ten foals were classified as affected by ENS, eight by PAS, whereas five foals suffered both the diseases. From admission until discharge or death, animals were constantly monitored by clinical, laboratory, and instrumental examinations, according to the severity of the disease. Each foal received proper treatments in relation to its specific clinical presentation; any resuscitation treatment (that could influence ET-1 concentrations) was recorded. Sick foals were sampled every 24 hours from the day of admission until discharge (n ¼ 12) or death (n ¼ 8, four with ENS, three with ENS and PAS, and one with PAS). The first sample was always collected before any medical treatment. Only 20 foals were repeatedly sampled during hospitalization, since three foals died soon after admission. Blood was collected in plain tubes and, immediately after clotting, serum was separated by centrifugation
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Fig. 1. Values of Big endothelin 1 recorded in six healthy foals during the first week of life. Boxes indicate the I to II interquartile interval, the horizontal line corresponds to the median, the vertical lines are the limits of outlier distribution according to the Tukey rule. Near outliers are indicated by the symbol “þ”. The same letter indicates values not statistically different; different letters correspond to P < 0.05.
(2500 g for 10 minutes) and stored at 20 C until analyses. 2.3. Endothelin 1 measurement Big ET-1 was measured in all the samples using a sandwich ELISA (IBL Hamburg). The kit includes a primary capturing antibody against the C-terminal 22 to 38 amino acid sequence of rat Big ET-1 which shows closer homology to the equine amino acid sequence [9] and a secondary antibody for detection, directed against a different antigenic site of the molecule. As a standard material to generate a calibration curve, human big ET-1 is included in the kit and the tetramethylbenzidine as a chromogen. The procedure was performed following the recommendation of the manufacturer of the kit, and the plates were read using an automated plate reader (Dasit multiscan, Dasit) at a wavelength of 450 nm. Intra-assay and interassay coefficients of variation reported by the manufacturer are lower than 5% and 13%, respectively at each value. However, to reduce the interassay variability, all samples were processed in the same batch. 2.4. Statistical analysis Statistical analysis was performed using Analyse-it software for Microsoft Excel. P values less than 0.05 were considered to be statistically significant. Results recorded in healthy foals at different sampling times were compared using a nonparametric ANOVA test for paired data (Friedman test) followed by the Wilcoxon signed-rank test to assess the difference between each time point. The
frequency of abnormal values (i.e., values higher than the maximum value recorded in healthy foals in the first 3 days of life) recorded at admission in all sick foals and in foals grouped on the basis of diagnosis was calculated. Finally, to achieve preliminary information about the prognostic value of ET-1, results recorded in nonsurviving foals at admission were compared with those of survivors using a nonparametric Student’s t test for unpaired samples (Mann Whitney U test). 3. Results All six healthy foals fulfilled the required characteristics, and in detail: birth weight ranging between 40 and 58 kg, mean Apgar index within 10 minutes of birth was 9 0.89, the time to stand up was 55.9 10.27 minutes and to the first suckle was 101 13.37 minutes. Results collected from healthy foals in the first 7 days of life are reported in Figure 1. As shown in the Figure 1, the serum concentration of Big ET-1 moderately increases in the first hours of life (17.4 4.0 pg/mL at 3 hours, 22.9 5.5 pg/mL at 12 hours) until 24 hours (20.4 6.4 pg/mL) after birth and then gradually decreases until Days 5, 6, and 7 (mean values around 12 pg/mL), when values are significantly lower than those recorded on Days 1, 2, and 3 (mean values around 18 pg/mL). No significant differences were recorded in the first 3 days of life. On the basis of lack of significant differences in healthy animals in the first five samples, values collected from sick foals at admission (before any medical treatment) were compared with the maximum value recorded in healthy
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foals from T0 to T4 (35.2 pg/mL). However, considering the low number of animals involved, only a descriptive analysis was performed. Such a comparison (Fig. 2) revealed that only 6 of 23 sick foals (26.1%) had values higher than this threshold. This group of foals with high Big ET-1 included 3 of 10 foals with ENS (30%), 2 of 8 foals with PAS (25%), and 1 of 5 foals with ENS and PAS (20%). Six of the sick foals received resuscitation treatment (four foals were treated with dobutamine and two foals with dobutamine and norepinephrine): two with PAS, one with ENS, and three with ENS and PAS. Eight sick foals (four with ENS, three with ENS and PAS, one with PAS) died during hospitalization, five of which received the previously mentioned treatments. Among these, only four (two with ENS, one with PAS, and one with both the diseases) had higher ET-1 values than controls at admission. Nevertheless, the results of nonsurviving foals (mean standard deviation: 23.5 5.7 pg/mL; median: 21.8 pg/mL; I to III interquartile range: 19.4–28.6 pg/mL) were not significantly different (P ¼ 0.123) from those of survivors (29.9 25.2 pg/mL, 18.6 pg/mL, 13.3–54 pg/mL, respectively). The analysis of sequential results (Fig. 3) showed that only one of eight nonsurviving foal had Big ET-1 values persistently below the maximum values recorded in healthy foals. Conversely, the serum concentrations of Big ET-1 remained persistently high or had only transient decreases in the four foals that at admission had abnormal values and increased during hospitalization in the three foals that had normal levels at admission. In surviving foals that had normal values of Big ET-1 at admission, instead, serum levels of Big ET-1 remained within the normal values during the whole hospitalization, whereas in the only one foal with high values at admission that was repeatedly sampled over time, Big ET-1 values rapidly normalized. 4. Discussion
Fig. 2. Distribution of values recorded in 23 sick foals at admission. The dotted gray line indicates the upper value recorded in clinically healthy foals aged less than 4 days. Big-ET-1, Big endothelin 1.
Despite the frequent occurrence of ENS and PAS in newborn foals, studies about prognostic markers of both these conditions are scarce. In human medicine, sepsis and asphyxia are known to induce increases in the serum concentration of ET-1 either in adults or in newborns, where increases of ET-1 or of its precursor Big ET-1 may provide prognostic information [6,7,30,31]. Conversely, no information is reported on ET-1 in foals affected by ENS and/or PAS and, more importantly, no information is available on the possible age-related changes of the serum concentration of this biomarker in healthy neonatal foals, so this represents the first study on the possible use of ET-1 for health status evaluation in newborn foals. Therefore, the first step of the study was focused on investigating the possible presence of changes in the serum concentration of Big ET-1 in the first days of life in healthy foals. Because, unfortunately, this part of the study was biased by the low number of animals, it was not possible to define appropriate reference intervals for each sampling time, using the approaches recommended by the current guidelines [32,33]. However, the statistical
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Fig. 3. Values recorded during hospitalization in sick foals. Foals with septicemia are indicated with the squares; foals with asphyxia are indicated with circles; foals with septicemia and asphyxia are indicated with the triangles. Black symbols indicate nonsurviving foals; white symbols indicate surviving foals. The dotted gray line indicates the upper value recorded in healthy foals less than 4 days old. Big ET-1, Big endothelin 1.
comparison of the results obtained in healthy foals in the first week of life revealed a trend that parallels what is reported in human newborns, where Big ET-1 values increase in the first hours of life and then start to decrease after 5 days until Day 30 [34,35]. A similar trend has been also reported in piglets [36]. In our caseload, even on Day 7, values were still higher than those reported in adult horses in studies that, however, used a different method for measuring Big ET-1 [17,18]. Because ET-1 is elevated in fetuses, where it contributes to maintain an adequate pulmonary vasodilation at birth followed by a progressive reduction of vascular resistance after birth [37], this agerelated change may depend on an incomplete removal of circulating ET-1 by the liver of newborns, less functional than in adults [31]. As already reported for other molecules involved in inflammation or tissue damages [38,39], independently from the mechanism responsible for these changes, the finding of higher serum concentration of Big ET-1 in newborn foals than in adults evidences the need to establish, in the future, age-specific reference intervals. This would avoid to erroneously interpreting as extremely high Big ET-1 values that actually depend on the young age of the foals. Results from sick foals, however, evidenced that the diagnostic or prognostic role of increased Big ET-1 levels at admission (before any medical treatment) seems to be moderate, because approximately one fourth (26.1%) of sick foals had values higher than the maximum value recorded in clinically healthy foals. Moreover, increases of serum Big ET-1 seem not to be associated with a specific condition because the group of foals with abnormal values included foals with ENS, PAS, or both. Additionally, although the majority of foals with abnormal levels of Big ET-1 (four of six) died during hospitalization, 4 of 8 nonsurviving foals had normal values at admission and no significant differences were found
between survivors and nonsurvivors. All these findings seem to contrast with what reported in human medicine, where high ET-1 values correlate with a poor prognosis either in sepsis or in conditions characterized by hypoxia [30,31,40]. Also, previous studies on adult horses reported that ET-1 may be a marker of severity of inflammatory conditions and negatively correlated with survival [17]. Therefore, it may be postulated that the magnitude of ET-1 responses to endotoxic shock or hypoxia in foals is less intense than in adult horses or in other species. Alternatively, the lack of differences between sick and healthy foals or between surviving and nonsurviving foals may depend on the short time elapsed between the onset of clinical signs and the first sampling or on different degree of severity of tissue damages in affected horses. Figueras-Aloy et al. [6] reported that the highest ET-1 levels in neonatal sepsis appeared at 3 to 5 days after the diagnosis and later decreased. Only newborns with severe sepsis presented a significant increase in ET-1 concentrations from the beginning of the septicemic process. However, any conclusion about the possible prognostic value of a single measurement of serum ET-1 at admission should be reviewed when age-specific reference intervals are established on a larger cohort of healthy neonatal foals. Conversely, this study reported that repeated measurement of Big ET-1 during hospitalization may be helpful in monitoring the course of the disease: Because of the heterogeneity of samplings collected during hospitalization (the different foals were admitted at different ages, foals received different therapies, such as resuscitation treatments, specific for their clinical conditions, and the length of hospitalization was variable), results recorded over time were not statistically compared to each other. However, with a single exception, Big ET-1 values in nonsurvivors, independently on the disease (PAS and/or ENS), remained over the maximum value recorded in healthy
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foals or, when normal at admission, increased over time. Five of the six animals that received resuscitation therapies did not survive; therefore, treatment was ineffective and may have contributed to the increase of Big ET-1 during hospitalization because it is known that in humans, during septic shock, the release or the production of endothelin may increase as a consequence of the infusion of catecholamines [41]. It must be stressed that the significance of high values in sequential samplings is stronger than the single value at admission. In healthy foals, in fact, the serum concentration of Big ET-1 decreases with age, and therefore, the difference between values recorded in healthy and in sick foals become even more evident as age increases. These results indicate that, when the disease progresses despite the administration of any kind of treatment, stimuli inducing the release of ET-1 from damaged endothelium persist and maintain the serum concentration of Big-ET-1 elevated.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
4.1. Conclusions [12]
In conclusion, this study evidenced that serum concentration of Big ET-1 in foals shares some similarity with that of human ET-1. In particular, age-related changes found in the first days of life are the same as recorded in human newborns. The low number of animals involved in the study is the most important limitation of the work to arrive to strong conclusions. However, considering that this is the first reference of using ET-1 for health status evaluation in foals and the promising results obtained, this work underlines the need of further investigations to create specific age-related reference ranges for the equine newborn. This information will be relevant on a diagnostic perspective, to avoid erroneously interpreting as high values that are actually normal in newborns. Moreover, because in nonsurviving foals, Big ET-1 remained over the maximum value recorded in clinically healthy horses or, when normal at admission, increased over time, this study suggested that repeated measurement of Big ET-1 during hospitalization may be helpful in monitoring the course of the disease. Prognostic information may be obtained by repeated analysis of Big ET-1 during hospitalization, but further studies are needed.
[13]
[14] [15] [16]
[17]
[18]
[19]
[20]
[21]
[22]
Competing Interests [23]
None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the article.
[24] [25]
References [1] Rubanyi GM, Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 1994;46:325–415. [2] Haynes WG. Endothelins as regulators of vascular tone in man. Clin Sci 1995;88:509–17. [3] Murohara T, Lefer AM. Autocrine effects of endothelin-1 on leukocyte-endothelial interaction: stimulation of endothelin B receptor subtype reduces endothelial adhesiveness via a nitric oxide dependent mechanism. Blood 1996;88:3894–900. [4] Finsnes F, Lyberg T, Christensen G, Skjønsberg OH. Effect of endothelin antagonism on the production of cytokines in eosinophilic
[26]
[27]
[28]
[29]
airway inflammation. Am J Physiol Lung Cell Mol Physiol 2001;280: L659–665. Ros J, Leivas A, Jiménez W, Morales M, Bosch-Marcé M, Arroyo V, et al. Effect of bacterial lipopolysaccharide on endothelin-1 production in human vascular endothelial cells. J Hepatol 1997; 26:81–7. Figueras-Aloy J, Gómez-Lopez L, Rodríguéz-Miguélez JM, JordánGarcía Y, Salvia-Roiges MD, Jiménez W, et al. Plasma endothelin-1 and clinical manifestations of neonatal sepsis. J Perinat Med 2004; 32:522–6. Kojima T, Isozaki-Fukuda Y, Takedatsu M, Ono A, Hirata Y, Kobayashi Y. Plasma endothelin-1 like immunoreactivity levels in neonates. Eur J Pediatr 1992;151:913–5. Abman SH. Recent advances in the pathogenesis and treatment of persistent pulmonary hypertension of the newborn. Neonatology 2007;91:283–90. Uchide T, Fujimori Y, Temma K, Sasaki T, Kizaki K, Hara Y, et al. cDNA cloning, sequence analysis and organ distribution of horse preproendothelin-2. J Cardiovasc Pharmacol 2004;44:S430–4. O’Sullivan ML, O’Grady MR, Minors SL. Plasma big endothelin-1, atrial natriuretic peptide, aldosterone, and norepinephrine concentrations in normal Doberman pinschers and Doberman pinschers with dilated cardiomyopathy. J Vet Int Med 2007;21:92–9. Prosek R, Sisson DD, Oyama MA, Biondo AW, Solter PE. Measurements of plasma endothelin immunoreactivity in healthy cats and cats with cardiomyopathy. J Vet Int Med 2004;18:826–30. Rossi G, Giordano A, Breda S, Lisi C, Roura X, Zatelli A, et al. Bigendothelin 1 (Big ET-1) and homocysteine in the serum of dogs with chronic kidney disease. Vet J 2013;198:109–15. Nakamura T, Kasai K, Sekiguchi Y, Banba N, Takahashi K, Hattori Y, et al. Elevation of plasma endothelin concentrations during endotoxin shock in dogs. Eur J Pharmacol 1991;205:277–82. Hood DM, Amoss MS, Grosenbaugh DA. Equine laminitis: a potential model of Raynaud’s phenomenon. Angiology 1990;41:270–7. Katwa LC, Johnson PJ, Ganjamt VK, Kreeger JM, Messer NT. Expression of endothelin in equine laminitis. Equine Vet J 1999;31:243–7. LeBlanc C, Moore RM. Plasma and synovial fluid endothelin-1 and nitric oxide concentrations in horses with and without joint disease. Am J Vet Res 2002;63:1648–54. Ramaswamy CM, Eades SC, Vegopa l CS, Hosgood GL, Garza F, Barker SA, et al. Plasma concentrations of endothelin-like immunoreactivity in healthy horses and horses with naturally acquired gastrointestinal tract disorders. Am J Vet Res 2002;63:454–9. Benamou AE, Art T, Marlin DJ, Roberts CA, Lekeux P. Variations in systemic and pulmonary endothelin-1 in horses with recurrent airway obstruction (heaves). Pulm Pharmacol Ther 1998; 11:231–5. Benamou AE, Marlin DJ, Lekeux P. Endothelin in the equine hypoxic pulmonary vasoconstrictive response to acute hypoxia. Equine Vet J 2001;33:345–53. Venugopal CS, Polikepahad S, Holmes EP, Heuvel JV, Leas TL, Moore RM. Endothelin receptor alterations in equine air way hyperreactivity. Can J Vet Res 2006;70:50–7. Galvin NP, Corley KTT. Causes of disease and death from birth to 12 months of age in the Thoroughbred horse in Ireland. Irish Vet J 2010;63:37–43. Wort SJ, Evans TW. The role of the endothelium in modulating vascular control in sepsis and related conditions. Br Med Bull 1999; 55:30–48. Lavoie JP, Madigan JE, Cullor JS, Powell WE. Haemodynamic, pathological, haematological and behavioural changes during endotoxin infusion in equine neonates. Equine Vet J 1990;22:23–9. Galvin N, Collins D. Perinatal asphyxia syndrome in the foal: review and a case report. Irish Vet J 2004;57:707–14. Wong D, Wilkins PA, Bain FT, Brockus C. Neonatal encephalopathy in foals. Compend Contin Educ Vet 2011;33:1–10. Rossdale PD, Ousey JC, Silver M, Fowden A. Studies on equine prematurity 6: guidelines for assessment of foal maturity. Equine Vet J 1984;16:300–2. Koterba AM. Physical examination. In: Koterba AM, Drummond WH, Kosch PC, editors. Equine clinical neonatology. Philadelphia, PA: Lea & Fabiger; 1990. p. 71–86. Brewer BD, Koterba AM. Development of a scoring system for the early diagnosis of equine neonatal sepsis. Equine Vet J 1988;20: 18–22. Knottenbelt DC, Holdstock N, Madigan JE, Knottenbelt DC, Holdstock N, Madigan JE. Neonatal syndromes. In: Equine neonatology: medicine and surgery. St. Louis, MO: Saunders Elsevier; 2004. p. 155–364.
A. Giordano et al. / Theriogenology 84 (2015) 667–673 [30] Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, et al. Definitions for sepsis and organ failure and guidelines for use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 1992;101:1644–55. [31] Paulus P, Jennewein C, Zacharowski K. Biomarkers of endothelial dysfunction: can they help us deciphering systemic inflammation and sepsis? Biomarkers 2011;16:S11–21. [32] Geffré A, Friedrichs K, Harr K, Concordet D, Trumel C, Braun JP. Reference values: a review. Vet Clin Pathol 2009;38:288–98. [33] Friedrichs KR, Harr KE, Freeman KP, Szladovits B, Walton RM, Barnhart KF, et al. American Society for Veterinary Clinical Pathology. ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics. Vet Clin Pathol 2012;41:441–53. [34] Nisell H, Hemsén A, Lunell NO, Wolff K, Lundberg MJ. Maternal and fetal levels of a novel polypeptide, endothelin: evidence for release during pregnancy and delivery. Gynecol Obstet Invest 1990;30:129–32. [35] Endo A, Shimada M, Ayusawa M, Minato M, Takada M, Takahashi S, et al. Nitric oxide and endothelin-1 during postnatal life. Biol Neonate 1996;70:15–20.
673
[36] Levy M, Tulloh RM, Komai H, Stuart-Smith K, Haworth SG. Maturation of the contractile response and its endothelial modulation in newborn porcine intrapulmonary arteries. Pediatr Res 1995;38:25–9. [37] Gao Y, Raj U. Regulation of the pulmonary circulation in the fetus and newborn. Physiol Rev 2010;90:1291–335. [38] Paltrinieri S, Giordano A, Villani M, Manfrin M, Panzani S, Veronesi MC. Influence of age and type of foaling plasma protein electrophoresis (PPE) and serum amyloid A (SAA) concentration and preliminary results on the role of PPE and SAA as markers of equine neonatal septicaemia. Vet J 2008;176:393–6. [39] Castagnetti C, Pirrone A, Mariella J, Mari G. Venous blood lactate evaluation in equine neonatal intensive care. Theriogenology 2010; 73:343–57. [40] Bennet J, Cooper D, Balakrishnan A, Rhodes M, Lewis M. Is the role for serum endothelin in predicting the severity of acute pancreatitis? Hepatobiliary Pancreat Dis Int 2006;5:290–3. [41] Voerman HJ, Stehouwer CD, van Kamp GJ, Strack van Schijndel RJ, Groeneveld AB, Thijs LG. Plasma endothelin levels are increased during septic shock. Crit Care Med 1992;20: 1097–101.