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Thyroid hormone concentrations in foals affected by perinatal asphyxia syndrome
Theriogenology 80 (2013) 624–629
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Thyroid hormone concentrations in foals affected by perinatal asphyxia syndrome Alessandro Pirrone a, *, Sara Panzani b, Nadia Govoni a, Carolina Castagnetti a, Maria Cristina Veronesi b a b
Department of Veterinary Medical Sciences, Università di Bologna, Bologna, Italy Department of Health, Animal Science and Food Safety, Faculty of Veterinary Medicine, Università degli Studi di Milano, Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2012 Received in revised form 3 June 2013 Accepted 4 June 2013
The hypothalamus-pituitary-thyroid axis has specific functions, mostly related to metabolic activities, cell differentiation, and development. To the authors’ knowledge, there are no studies about thyroid hormone (TH) concentrations in foals affected by perinatal asphyxia syndrome (PAS). Hence, the aims of the study are (1) to evaluate plasma TH concentrations (T3 and T4) in healthy foals during the first 7 days of life; (2) to evaluate plasma TH concentration (T3 and T4) in critically ill foals affected by PAS during the first 7 days of hospitalization; and (3) to compare TH concentrations between surviving and nonsurviving critically ill foals. Forty-five Standardbred foals were enrolled in this prospective observational study: 21 healthy foals (group 1) and 24 foals affected by PAS (group 2). Jugular blood samples were collected within 10 minutes from birth/admission and every 24 hours for 7 days (t0–t7). TH concentrations were analyzed by RIA. In both groups, T3 concentration was significantly lower at t4, t5, t6, and t7 compared with t1 (P < 0.05), and T4 concentration was significantly higher at birth than at all other time points (P < 0.01). No differences were found in TH concentrations at admission between surviving (n ¼ 20) and nonsurviving (n ¼ 4) foals. Statistical comparison between healthy and PAS foals divided into age groups showed significantly lower TH concentrations at t0 in PAS foals <12 hours old at admission (P < 0.01). In conclusion, PAS may cause lower T3 and T4 concentrations in affected foals than in age-matched healthy foals, as reported for other systemic illnesses, such as sepsis and prematurity. TH concentrations showed no prognostic value, which maybe due to the small number of nonsurviving foals in this study. Further studies are needed to find out if thyroid replacement therapy could be useful in the treatment of critically ill foals affected by PAS. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Foal Thyroid hormones Perinatal asphyxia syndrome
1. Introduction The hypothalamus-pituitary-thyroid (HPT) axis has specific functions, mostly related to metabolic activities, cell differentiation, and development [1]. In addition to its effects on energy metabolism, thyroid hormones (THs) are essential for both prenatal and postnatal developmental events including organ formation and skeletal maturation [2]. * Corresponding author. Tel.: þ390512097587; fax: þ390512097568. E-mail address: [email protected] (A. Pirrone). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.06.003
In healthy full-term human neonates at birth, Glinoer et al. [3] reported normal free T4 and low free T3 concentrations compared with adults; thyroid-stimulating hormone (TSH) consistently increased during the first 24 hours of life, and an abrupt rise of T3 and T4 concentrations was observed. Kratzsch and Pulzer [4] reported that T4 feedback inhibition caused a TSH decrease from Day 3 or 4 of age. In newborn foals, THs are essential for normal organ development and growth, and, as it might be expected, deficiencies result in more significant clinical problems in foals than in adults [5]. In horses, serum TH
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concentrations are much higher in neonates than adults, slowly decreasing to adult concentrations over the first weeks of life [6–8]. Premature human babies have an immature HPT axis, and they show lower T4 levels than full-term neonates; T4 concentration is correlated with gestational age and birth weight [9]. Septicemia is also indicated as a cause of low TH concentrations in newborn babies [10]. Low TH levels in human babies are also correlated with respiratory distress syndrome [11] and asphyxia [12]. Perinatal asphyxia triggers several systemic alterations, including a rapid increase in the concentration of some hormones [13,14], but few studies have been conducted on the effect of perinatal asphyxia on TH concentrations [12,15]. The more recent study found significantly lower levels of TSH, T4, and T3 in asphyxiated human newborns compared with controls, suggesting a central hypothyroidism secondary to asphyxia [15]. Low TH levels have been measured also in sick foals with noncritical conditions [16–19]. Irvine [20] suggested that the decrease in metabolism reported in sick foals, which results in inadequate thermogenesis and lethargy, may be due to a circulating TH deficiency; furthermore, the severity of the symptoms was related to the severity of hormone deficiency. Dysfunction of the thyroid gland has been reported in foals born from mares grazing endophyte-infected fescue [19] and in foals with congenital hypothyroidism/goiter [17,21]. In a recent study, a lower concentration of TH was found in sick foals affected by varying pathologies compared with healthy foals [22]. Information about the HPT axis and TH concentrations in critically ill foals is lacking. Total T4, total T3, free T4, and free T3 levels were lower in septic foals when compared with sick nonseptic foals and healthy foals, and these concentrations were even lower in nonsurviving septic foals [23]. Similar results were reported in premature foals, which had low TH levels and an exaggerated TSH response to TRH [8,24]. To our knowledge, there are no studies about TH concentrations in foals affected by perinatal asphyxia syndrome (PAS). This syndrome is a relatively common neonatal disorder that can result from any event that impairs oxygen delivery to cells occurring prepartum, intrapartum, or during the early neonatal period. Asphyxia triggers varying degrees of multisystemic effects, with renal, gastrointestinal, cardiopulmonary, endocrine, and neurologic symptoms [25]. Despite the clinical relevance of this syndrome, most information regarding the pathophysiology of this disease is directed at infants, and equine-specific information is exceedingly sparse [26]. Hence, the aims of the study are (1) to evaluate plasma TH concentrations (T3 and T4) in healthy foals during the first 7 days of life; (2) to evaluate plasma TH concentrations (T3 and T4) in critically ill foals affected by PAS during the first 7 days of hospitalization; and (3) to compare TH concentrations between surviving and nonsurviving critically ill foals. We hypothesize that TH concentrations will be lower in PAS foals than in age-matched healthy foals and that survivors will show higher TH concentrations than nonsurvivors.
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2. Materials and methods 2.1. Animals Forty-five Standardbred foals, born during the 2010 and 2011 breeding seasons, were enrolled in this prospective observational study: 21 healthy foals (group 1) and 24 foals affected by PAS (group 2). Foals included in group 1 were born on a Standardbred breeding farm in Northern Italy. The foals were classified as healthy when they had an Apgar score 9 [27], a normal clinical evaluation during the period of study, including a complete blood count and serum biochemistry at birth and an IgG serum concentration 800 mg/dL at 18 to 24 hours of life. In clinically healthy foals, jugular venous samples for TH measurements were obtained within 10 minutes of birth (t0) and every 24 hours until 7 days of life (t1–t7). Blood was collected into heparinized plastic vials (S-Monovette; Sarstedt), and the sample was delivered to the laboratory within 30 minutes of collection. After centrifugation at 2200 g for 10 minutes, all samples were stored at 20 C and analyzed within 2 months after collection. Foals included in group 2 were referred to the neonatal intensive care unit after birth. The inclusion criterion for group 2 was the diagnosis of PAS requiring level 2 or 3 of intensive care on the basis of the classification proposed by Koterba [28]. Level 2 care is provided to neonates that are quite severely affected; foals may be unable to stand or unable to nurse from the mare and need round-the-clock care; this level of care usually involves separation of the foal from the dam. Level 3 care is intended for extremely compromised foals that usually have multisystem dysfunction, need round-the-clock care, and must be assisted by specialists. Foals were classified as affected by PAS on the basis of history and clinical signs, especially those of neurologic dysfunction [29] and exclusion of other neurologic diseases such as meningitis or trauma. Typical historical events included dystocia, red bag, or avillous placenta, and common clinical signs included loss or absence of the suck reflex, inappropriate teat-seeking behavior, dysphagia, seizures, hyperreactivity, and weakness associated with an elevated serum creatinine concentration at less than 24 hours of age [30]. Foals affected by PAS and other pathologies (i.e., sepsis, prematurity, neonatal isoerythrolysis) were excluded from the study. All foals of group 2 received a complete and standardized clinical evaluation at admission. Venous jugular blood was also collected for hematobiochemical evaluation and for blood culture. Serum IgG concentration was measured when the foals were at least 18 hours of age (SNAP Foal, IDEXX, Milano, Italy). In group 2, plasma TH concentrations were measured at admission (t0) and every 24 hours during the first 7 days of hospitalization (t1–t7). Blood was collected into heparinized plastic vials (S-Monovette; Sarstedt), and the sample was delivered to the laboratory within 30 minutes of collection. After centrifugation at 2200 g for 10 minutes, all samples were stored at 20 C and analyzed within 2 months after collection.
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Table 1 Means SD of plasma T3 and T4 concentrations (nmol/L) in healthy foals (group 1) from birth to 7 days of life. TH
t0
t1
t2
t3
t4
t5
t6
t7
T3 T4
9.63a,b 4.76 394.81a 140.8
11.00b 2.76 249.5b 82.73
10.88b,c 2.53 210.85b 47.20
9.06b,c 3.50 178.91b 73.96
7.86a,c 1.72 146.78b 35.31
7.01a,c 1.18 116.35b 34.27
6.62a,c 1.24 81.68b 30.43
7.03a,c 2.42 100.67b 79.68
a,b,c
Different superscript letters in rows indicate significant differences between time points with GLM analysis (P < 0.01).
All procedures on the animals were carried out with the approval of the Ethical Committee of the Faculty of Veterinary Medicine, University of Bologna, in accordance with DL 116/92, approved by the Ministry of Health. Oral informed consent was given by the owners.
A P value <0.05 was considered statistically significant. All analyses were carried out using a commercial software (SPSS, version 13.0, SPSS Inc., Chicago, IL, USA).
2.2. TH analysis
Totally, 8 fillies and 13 colts were enrolled in group 1. An age-specific normal range for plasma TH (T3 and T4) concentrations was calculated from data collected and it is shown in Table 1. The GLM found a significant change over time within the subjects in plasma T3 concentration (P < 0.01). Post-hoc test showed a nonstatistically significant increasing trend at t1 compared with t0 and significantly lower concentrations at t4, t5, t6, and t7 compared with t1 (P < 0.01). In plasma T4 concentration, GLM found a significant change over time within the subjects, and post-hoc test showed a significantly higher concentration at birth than at all other time points (P < 0.01). In group 2, the median age of the study population at admission was 22 hours (0–48 hours). There were 6 fillies (6 survivors) and 18 colts (14 survivors and 4 nonsurvivors). The results of the statistical analysis of time-dependent plasma TH changes in group 2, measured during the first 7 days of hospitalization, are reported in Table 2. In group 2, the GLM found a significant change over time within the subjects in plasma T3 concentration (P < 0.01). Post-hoc test showed a nonstatistically significant decreasing trend from t0 to t4 and significantly lower concentrations at t4, t5, t6, and t7 compared with t1 (P < 0.01). In plasma T4 concentration, GLM found a significant change over time within the subjects, and post-hoc test showed a significantly higher concentration at admission than at all other time points (P < 0.01). At admission, the mean plasma T3 concentration in surviving foals (n ¼ 20) was 6.57 4.67 nmol/L and the mean plasma T4 concentration was 259.79 60.97 nmol/L; the mean plasma T3 concentration in nonsurviving foals (n ¼ 4) was 6.22 4.33 nmol/L and the mean plasma T4 concentration was 263.64 67.00 nmol/L. No statistically significant difference was found in plasma T3 and T4 concentrations at admission between surviving and nonsurviving foals (P ¼ 0.081).
TH (T3 and T4) plasma concentrations were analyzed by RIA using commercial kits supplied by Izotop (Institute of Isotopes Co., Ltd., Budapest). The T3 antibody crossreacted with 3,30 -diiodo-L-thyronine (<1.9%), T4 (<0.06%), and reverse T3 (rT3) (<0.016%). The T4 antibody crossreacted with T3 (<12.6%), rT3 (<0.89%), and 3,30 -diiodoL-thyronine (0.11%). In order to determine the agreement between T3 and T4 standards and endogenous hormones in foals’ plasma, a pooled sample containing high TH concentrations was serially diluted (1:1–1:8) with RIA buffer. A regression analysis was used to determine the agreement between standards and endogenous hormones run in the same assay. A high degree of agreement was confirmed by regression test (r2 ¼ 0.98, P < 0.01), demonstrating the specificity of the procedure to determine TH concentrations in foals. Assay sensitivity was 0.22 and 14.53 nmol/L for T3 and T4, respectively. Intra- and interassay coefficients of variation were 2.64% and 11.00% for T3 and 4.64% and 12.16% for T4, respectively. Values are expressed as nmol/L. 2.3. Statistical analysis A general linear model (GLM) for repeated measurements with Bonferroni’s post-hoc comparisons was used to evaluate the T3 and T4 concentrations over time in healthy foals. The same method was used to evaluate time-dependent changes from t0 to t7 in foals affected by PAS. The Mann-Whitney U-test was performed to compare T3 and T4 values at admission in critically ill foals divided into subgroups on the basis of their age at admission (0–12, 13–36, and 37–60 hours) with healthy age-matched foals. The Mann-Whitney U-test was used to compare TH concentrations of surviving and nonsurviving foals at admission (t0), without any correction for age.
3. Results
Table 2 Means SD of plasma T3 and T4 concentrations (nmol/L) in foals affected by PAS (group 2) from admission to 7 days of hospitalization. TH
t0
t1
t2
t3
t4
t5
t6
t7
T3 T4
6.57a,b 4.67 259.79a 60.97
5.47b 2.27 188.87b 55.78
4.29b,c 2.02 162.89b 46.75
3.97b,c 1.83 141.85b 41.03
3.89a,c 1.57 108.42b 16.57
3.48a,c 1.16 101.54b 16.32
3.35a,c 1.22 71.49b 17.79
3.76a,c 1.01 58.78b 15.72
a,b,c
Different superscript letters in rows indicate significant differences between time points with GLM analysis (P <0.01).
A. Pirrone et al. / Theriogenology 80 (2013) 624–629
Statistical comparison between group 1 and group 2 foals divided into age groups showed significantly lower plasma T3 and T4 concentrations only in PAS foals (n ¼ 11) less than 12 hours old at admission (P < 0.01) (Table 3). 4. Discussion To the authors’ knowledge, this is the first study in which plasma TH concentrations (T3 and T4) were evaluated in neonatal foals affected by PAS, in comparison with a control group of clinically healthy foals. In healthy foals, this study showed a peak of plasma T3 concentration at 24 hours of age; it remained high for up to 48 hours and then showed a decreasing trend for the rest of the study period. These results are in agreement with the previous studies performed in neonatal foals [6,20], which showed plasma T3 concentrations seven times higher in neonates than in adults, with a progressive increase in the first 10 hours of life, peaking at 24 to 48 hours, and followed by a progressive decrease. However, this result disagrees with the data found by Panzani et al. [22], who reported a time-dependent decrease from birth in the first 2 weeks of life. On the other hand, plasma thyroxine concentration was high at birth in healthy foals and decreased significantly from as early as 24 hours of life. These data are partially in agreement with that reported by Panzani et al. [22], who found high T4 plasma levels at birth, followed by a significant decrease at 12 hours. Some studies reported plasma T4 concentrations at birth 14 times higher than in adults, with a subsequent rapid decrease to adult concentrations at 16 days of life [6,20]. A large standard deviation was found at t0, as previously reported by other authors [22,23]. Such a high TH concentration has been detected only in newborn foals, and they are higher than in any other species in any physiological stage. The high TH concentrations of newborn foals may be responsible for their high thermogenic capacity and remarkable rapidity of growth during the perinatal period, especially of musculo-skeletal and nervous systems [6]. Conversely, in human babies born at term, a further increase of both plasma T3 and T4 concentrations in the early hours of life has been reported [31]. Fisher et al. [32] hypothesized that this increase is due to a shift from a predominantly inactive thyroid gland to a state of hyperactivity, which allows an adequate adaptation of the newborn to the extrauterine environment. Moreover, the Table 3 Plasma TH concentration (nmol/L) in healthy foals (group 1) divided into three subgroups on the basis of age and in PAS foals (group 2) divided into three subgroups on the basis of age at admission. Foals
Number of samples
T3
Healthy (t0) PAS (0–12 h) Healthy (t1) PAS (13–36 h) Healthy (t2) PAS (37–60 h)
21 11 21 8 21 5
9.63 3.95 11.00 8.64 10.88 9.01
T4
4.76a 2.17b 2.76a 5.12a 2.53a 5.72a
394.81 242.64 249.5 288.97 210.85 250.86
140.8a 61.85b 82.73a 52.94a 47.20a 65.55a
a,b Different superscript letters in columns indicate significant differences with Mann-Whitney U-test (P < 0.01).
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increase in the concentrations of T4-binding globulins could be a further cause of total T4 increase [33]. In infants, the temperature variation between intrauterine and extrauterine environment appears to be able to stimulate the HPT axis, with an increased TRH and TSH secretion immediately after birth; this increase has a brief duration, with a peak at about 30 minutes after birth, followed by a progressive decrease in plasma TSH concentration at 3 to 5 days of life due to the T4 negative feedback [32]. In the present study, the monitoring of TH concentrations in foals affected by PAS showed a significant timedependent change from the fourth day of hospitalization in T3 concentrations and from 24 hours of hospitalization in T4 concentrations. It is worth noting the absence of the plasma T3 increase at t1 reported in healthy foals in the present study. The results of group 2 cannot be compared with those of other authors, because, to the authors’ knowledge, there is no study describing TH concentrations in foals affected by PAS. TH concentrations in sick foals were investigated by several authors. Silver et al. [24] evaluated T3 concentration in premature foals finding lower concentrations than in term foals. In a recent study of 26 sick foals with varying diagnoses (12 affected by PAS), Panzani et al. [22] reported lower plasma T3 concentrations than in healthy foals during the first week of life and lower plasma T4 concentrations during the first 3 days of life. In a study by Himler et al. [23], lower TH concentrations were observed in foals admitted to the ICU compared with healthy foals aged 12 to 36 hours, except for reverse T3. These authors reported that both the presence of disease and prematurity were associated with TH deficiencies, because of the lower values found in premature sick foals compared with term sick foals. The authors further suggested that the HPT axis immaturity of premature foals could aggravate the symptoms in animals debilitated by systemic illness. In human medicine, T4 concentration was lower in premature than in full-term babies due to the immaturity of the HPT axis, and the concentration was related to gestational age and birth weight [9]. Newborn human babies with respiratory distress syndrome had normal TSH, T3, T4, and fT4 concentrations at birth, and a significant decrease in T3, T4, and fT4 on the fifth day of life; TSH values remained rather normal [34]. The same data have been reported by Pereira and Procianoy [35] at 18 to 24 hours of life in infants undergoing a period of perinatal asphyxia. In another study, the same authors investigated the effects of perinatal asphyxia on THs concentrations, and they found statistically lower values in babies with moderate or severe hypoxic-ischemic encephalopathy compared with healthy ones [15]. In the present study, TH concentrations observed in PAS foals <12 hours old at admission were significantly lower than in healthy foals within 10 minutes from birth. This result is only partially comparable with those of other authors. Panzani et al. [22] found a similar result in premature, PAS, and septicemic foals compared with healthy foals, although TH concentrations were evaluated on the basis of foal’s age at each sampling time, regardless of the elapsed time from admission. A similar result was also obtained in sick foals (septic and nonseptic) by Himler et al.
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[23], who assessed TH concentrations only at admission independently of age and did not assess age-related changes in TH concentrations. As suggested by Himler et al. [23], in sick nonseptic and septic foals, the lower TH concentrations in foals affected by PAS found in this study could be the expression of nonthyroidal illness syndrome (NTIS or euthyroid sick syndrome). This is a well-recognized syndrome described in adult dogs and humans characterized by low TH concentrations in patients with severe nonthyroidal illness [36–40]. This syndrome is characterized by low T3 and elevated rT3 concentrations, associated with normal or low T4 and TSH concentrations [40]. NTIS probably represents an adaptive response to a systemic illness with a suppressive effect on the HPT axis and a decreased metabolism preventing organ dysfunction or death [36]. Initially, the conversion of T4 to T3 in peripheral tissues decreases, and, as the severity of illness, and often the associated starvation, progresses, T4 concentration also decreases, suggesting dysfunction at the hypothalamic, pituitary, or thyroid gland level [37]. As reported by Breuhaus [5], NTIS can also result in decreased TH-binding proteins and decreased binding protein affinity and capacity. This effect of illness decreases the concentrations of total T4 and T3, but has less effect on free fraction concentrations. Other causes of NTIS may be represented by circulating cytokines generated during inflammation or treatments with some medications such as glucocorticoids or amiodarone [41,42]. Van der Poll et al. [41] found that tumor necrosis factor is involved, either directly or indirectly, in the pathogenesis of this syndrome. Moreover, in healthy individuals injected with interferon-a, TSH and T3 decrease while reverse T3 increases. This biochemical picture is similar to euthyroid sick syndrome and may be mediated through interleukin-6 [42]. It is worth noting that hypoxia can also activate specific mediators, such as cytokines [26]. Aly et al. [43] found the concentrations of IL-1b, IL-6, and tumor necrosis factor-a in cerebrospinal fluid and serum significantly higher in human neonates with hypoxic-ischemic encephalopathy when compared with infants in the control group. Moreover, all three cytokines in the cerebrospinal fluid correlated significantly with the clinical severity of asphyxia. We suggest that cytokine increase during perinatal asphyxia could trigger the effects on the neonatal HPT axis causing the lower TH concentrations found in this study in PAS foals <12 hours old. Because assessment of NTIS was not a specific aim of the present study, plasma rT3 concentration was not evaluated; therefore, we do not have enough data to confirm that foals affected by PAS had NTIS. In the present study, a prognostic value of TH concentrations at admission was not found, probably due to the low number of nonsurviving foals (n ¼ 4); therefore, it warrants further investigations on a larger number of animals. A prognostic value of TH concentrations has been found by Himler et al. [23], with lower TH concentrations in nonsurviving premature and septic foals than in surviving premature and septic foals. Data obtained in this trial suggest, therefore, that PAS may cause lower T3 and T4 concentrations in affected foals than in age-matched healthy foals, as reported for other
systemic illnesses, such as sepsis and prematurity. Further studies are needed to find out if thyroid replacement therapy could be useful in the treatment of critically ill foals affected by PAS.
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Thyroid hormone concentrations in foals affected by perinatal asphyxia syndrome Alessandro Pirrone a, *, Sara Panzani b, Nadia Govoni a, Carolina Castagnetti a, Maria Cristina Veronesi b a b
Department of Veterinary Medical Sciences, Università di Bologna, Bologna, Italy Department of Health, Animal Science and Food Safety, Faculty of Veterinary Medicine, Università degli Studi di Milano, Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2012 Received in revised form 3 June 2013 Accepted 4 June 2013
The hypothalamus-pituitary-thyroid axis has specific functions, mostly related to metabolic activities, cell differentiation, and development. To the authors’ knowledge, there are no studies about thyroid hormone (TH) concentrations in foals affected by perinatal asphyxia syndrome (PAS). Hence, the aims of the study are (1) to evaluate plasma TH concentrations (T3 and T4) in healthy foals during the first 7 days of life; (2) to evaluate plasma TH concentration (T3 and T4) in critically ill foals affected by PAS during the first 7 days of hospitalization; and (3) to compare TH concentrations between surviving and nonsurviving critically ill foals. Forty-five Standardbred foals were enrolled in this prospective observational study: 21 healthy foals (group 1) and 24 foals affected by PAS (group 2). Jugular blood samples were collected within 10 minutes from birth/admission and every 24 hours for 7 days (t0–t7). TH concentrations were analyzed by RIA. In both groups, T3 concentration was significantly lower at t4, t5, t6, and t7 compared with t1 (P < 0.05), and T4 concentration was significantly higher at birth than at all other time points (P < 0.01). No differences were found in TH concentrations at admission between surviving (n ¼ 20) and nonsurviving (n ¼ 4) foals. Statistical comparison between healthy and PAS foals divided into age groups showed significantly lower TH concentrations at t0 in PAS foals <12 hours old at admission (P < 0.01). In conclusion, PAS may cause lower T3 and T4 concentrations in affected foals than in age-matched healthy foals, as reported for other systemic illnesses, such as sepsis and prematurity. TH concentrations showed no prognostic value, which maybe due to the small number of nonsurviving foals in this study. Further studies are needed to find out if thyroid replacement therapy could be useful in the treatment of critically ill foals affected by PAS. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Foal Thyroid hormones Perinatal asphyxia syndrome
1. Introduction The hypothalamus-pituitary-thyroid (HPT) axis has specific functions, mostly related to metabolic activities, cell differentiation, and development [1]. In addition to its effects on energy metabolism, thyroid hormones (THs) are essential for both prenatal and postnatal developmental events including organ formation and skeletal maturation [2]. * Corresponding author. Tel.: þ390512097587; fax: þ390512097568. E-mail address: [email protected] (A. Pirrone). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.06.003
In healthy full-term human neonates at birth, Glinoer et al. [3] reported normal free T4 and low free T3 concentrations compared with adults; thyroid-stimulating hormone (TSH) consistently increased during the first 24 hours of life, and an abrupt rise of T3 and T4 concentrations was observed. Kratzsch and Pulzer [4] reported that T4 feedback inhibition caused a TSH decrease from Day 3 or 4 of age. In newborn foals, THs are essential for normal organ development and growth, and, as it might be expected, deficiencies result in more significant clinical problems in foals than in adults [5]. In horses, serum TH
A. Pirrone et al. / Theriogenology 80 (2013) 624–629
concentrations are much higher in neonates than adults, slowly decreasing to adult concentrations over the first weeks of life [6–8]. Premature human babies have an immature HPT axis, and they show lower T4 levels than full-term neonates; T4 concentration is correlated with gestational age and birth weight [9]. Septicemia is also indicated as a cause of low TH concentrations in newborn babies [10]. Low TH levels in human babies are also correlated with respiratory distress syndrome [11] and asphyxia [12]. Perinatal asphyxia triggers several systemic alterations, including a rapid increase in the concentration of some hormones [13,14], but few studies have been conducted on the effect of perinatal asphyxia on TH concentrations [12,15]. The more recent study found significantly lower levels of TSH, T4, and T3 in asphyxiated human newborns compared with controls, suggesting a central hypothyroidism secondary to asphyxia [15]. Low TH levels have been measured also in sick foals with noncritical conditions [16–19]. Irvine [20] suggested that the decrease in metabolism reported in sick foals, which results in inadequate thermogenesis and lethargy, may be due to a circulating TH deficiency; furthermore, the severity of the symptoms was related to the severity of hormone deficiency. Dysfunction of the thyroid gland has been reported in foals born from mares grazing endophyte-infected fescue [19] and in foals with congenital hypothyroidism/goiter [17,21]. In a recent study, a lower concentration of TH was found in sick foals affected by varying pathologies compared with healthy foals [22]. Information about the HPT axis and TH concentrations in critically ill foals is lacking. Total T4, total T3, free T4, and free T3 levels were lower in septic foals when compared with sick nonseptic foals and healthy foals, and these concentrations were even lower in nonsurviving septic foals [23]. Similar results were reported in premature foals, which had low TH levels and an exaggerated TSH response to TRH [8,24]. To our knowledge, there are no studies about TH concentrations in foals affected by perinatal asphyxia syndrome (PAS). This syndrome is a relatively common neonatal disorder that can result from any event that impairs oxygen delivery to cells occurring prepartum, intrapartum, or during the early neonatal period. Asphyxia triggers varying degrees of multisystemic effects, with renal, gastrointestinal, cardiopulmonary, endocrine, and neurologic symptoms [25]. Despite the clinical relevance of this syndrome, most information regarding the pathophysiology of this disease is directed at infants, and equine-specific information is exceedingly sparse [26]. Hence, the aims of the study are (1) to evaluate plasma TH concentrations (T3 and T4) in healthy foals during the first 7 days of life; (2) to evaluate plasma TH concentrations (T3 and T4) in critically ill foals affected by PAS during the first 7 days of hospitalization; and (3) to compare TH concentrations between surviving and nonsurviving critically ill foals. We hypothesize that TH concentrations will be lower in PAS foals than in age-matched healthy foals and that survivors will show higher TH concentrations than nonsurvivors.
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2. Materials and methods 2.1. Animals Forty-five Standardbred foals, born during the 2010 and 2011 breeding seasons, were enrolled in this prospective observational study: 21 healthy foals (group 1) and 24 foals affected by PAS (group 2). Foals included in group 1 were born on a Standardbred breeding farm in Northern Italy. The foals were classified as healthy when they had an Apgar score 9 [27], a normal clinical evaluation during the period of study, including a complete blood count and serum biochemistry at birth and an IgG serum concentration 800 mg/dL at 18 to 24 hours of life. In clinically healthy foals, jugular venous samples for TH measurements were obtained within 10 minutes of birth (t0) and every 24 hours until 7 days of life (t1–t7). Blood was collected into heparinized plastic vials (S-Monovette; Sarstedt), and the sample was delivered to the laboratory within 30 minutes of collection. After centrifugation at 2200 g for 10 minutes, all samples were stored at 20 C and analyzed within 2 months after collection. Foals included in group 2 were referred to the neonatal intensive care unit after birth. The inclusion criterion for group 2 was the diagnosis of PAS requiring level 2 or 3 of intensive care on the basis of the classification proposed by Koterba [28]. Level 2 care is provided to neonates that are quite severely affected; foals may be unable to stand or unable to nurse from the mare and need round-the-clock care; this level of care usually involves separation of the foal from the dam. Level 3 care is intended for extremely compromised foals that usually have multisystem dysfunction, need round-the-clock care, and must be assisted by specialists. Foals were classified as affected by PAS on the basis of history and clinical signs, especially those of neurologic dysfunction [29] and exclusion of other neurologic diseases such as meningitis or trauma. Typical historical events included dystocia, red bag, or avillous placenta, and common clinical signs included loss or absence of the suck reflex, inappropriate teat-seeking behavior, dysphagia, seizures, hyperreactivity, and weakness associated with an elevated serum creatinine concentration at less than 24 hours of age [30]. Foals affected by PAS and other pathologies (i.e., sepsis, prematurity, neonatal isoerythrolysis) were excluded from the study. All foals of group 2 received a complete and standardized clinical evaluation at admission. Venous jugular blood was also collected for hematobiochemical evaluation and for blood culture. Serum IgG concentration was measured when the foals were at least 18 hours of age (SNAP Foal, IDEXX, Milano, Italy). In group 2, plasma TH concentrations were measured at admission (t0) and every 24 hours during the first 7 days of hospitalization (t1–t7). Blood was collected into heparinized plastic vials (S-Monovette; Sarstedt), and the sample was delivered to the laboratory within 30 minutes of collection. After centrifugation at 2200 g for 10 minutes, all samples were stored at 20 C and analyzed within 2 months after collection.
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A. Pirrone et al. / Theriogenology 80 (2013) 624–629
Table 1 Means SD of plasma T3 and T4 concentrations (nmol/L) in healthy foals (group 1) from birth to 7 days of life. TH
t0
t1
t2
t3
t4
t5
t6
t7
T3 T4
9.63a,b 4.76 394.81a 140.8
11.00b 2.76 249.5b 82.73
10.88b,c 2.53 210.85b 47.20
9.06b,c 3.50 178.91b 73.96
7.86a,c 1.72 146.78b 35.31
7.01a,c 1.18 116.35b 34.27
6.62a,c 1.24 81.68b 30.43
7.03a,c 2.42 100.67b 79.68
a,b,c
Different superscript letters in rows indicate significant differences between time points with GLM analysis (P < 0.01).
All procedures on the animals were carried out with the approval of the Ethical Committee of the Faculty of Veterinary Medicine, University of Bologna, in accordance with DL 116/92, approved by the Ministry of Health. Oral informed consent was given by the owners.
A P value <0.05 was considered statistically significant. All analyses were carried out using a commercial software (SPSS, version 13.0, SPSS Inc., Chicago, IL, USA).
2.2. TH analysis
Totally, 8 fillies and 13 colts were enrolled in group 1. An age-specific normal range for plasma TH (T3 and T4) concentrations was calculated from data collected and it is shown in Table 1. The GLM found a significant change over time within the subjects in plasma T3 concentration (P < 0.01). Post-hoc test showed a nonstatistically significant increasing trend at t1 compared with t0 and significantly lower concentrations at t4, t5, t6, and t7 compared with t1 (P < 0.01). In plasma T4 concentration, GLM found a significant change over time within the subjects, and post-hoc test showed a significantly higher concentration at birth than at all other time points (P < 0.01). In group 2, the median age of the study population at admission was 22 hours (0–48 hours). There were 6 fillies (6 survivors) and 18 colts (14 survivors and 4 nonsurvivors). The results of the statistical analysis of time-dependent plasma TH changes in group 2, measured during the first 7 days of hospitalization, are reported in Table 2. In group 2, the GLM found a significant change over time within the subjects in plasma T3 concentration (P < 0.01). Post-hoc test showed a nonstatistically significant decreasing trend from t0 to t4 and significantly lower concentrations at t4, t5, t6, and t7 compared with t1 (P < 0.01). In plasma T4 concentration, GLM found a significant change over time within the subjects, and post-hoc test showed a significantly higher concentration at admission than at all other time points (P < 0.01). At admission, the mean plasma T3 concentration in surviving foals (n ¼ 20) was 6.57 4.67 nmol/L and the mean plasma T4 concentration was 259.79 60.97 nmol/L; the mean plasma T3 concentration in nonsurviving foals (n ¼ 4) was 6.22 4.33 nmol/L and the mean plasma T4 concentration was 263.64 67.00 nmol/L. No statistically significant difference was found in plasma T3 and T4 concentrations at admission between surviving and nonsurviving foals (P ¼ 0.081).
TH (T3 and T4) plasma concentrations were analyzed by RIA using commercial kits supplied by Izotop (Institute of Isotopes Co., Ltd., Budapest). The T3 antibody crossreacted with 3,30 -diiodo-L-thyronine (<1.9%), T4 (<0.06%), and reverse T3 (rT3) (<0.016%). The T4 antibody crossreacted with T3 (<12.6%), rT3 (<0.89%), and 3,30 -diiodoL-thyronine (0.11%). In order to determine the agreement between T3 and T4 standards and endogenous hormones in foals’ plasma, a pooled sample containing high TH concentrations was serially diluted (1:1–1:8) with RIA buffer. A regression analysis was used to determine the agreement between standards and endogenous hormones run in the same assay. A high degree of agreement was confirmed by regression test (r2 ¼ 0.98, P < 0.01), demonstrating the specificity of the procedure to determine TH concentrations in foals. Assay sensitivity was 0.22 and 14.53 nmol/L for T3 and T4, respectively. Intra- and interassay coefficients of variation were 2.64% and 11.00% for T3 and 4.64% and 12.16% for T4, respectively. Values are expressed as nmol/L. 2.3. Statistical analysis A general linear model (GLM) for repeated measurements with Bonferroni’s post-hoc comparisons was used to evaluate the T3 and T4 concentrations over time in healthy foals. The same method was used to evaluate time-dependent changes from t0 to t7 in foals affected by PAS. The Mann-Whitney U-test was performed to compare T3 and T4 values at admission in critically ill foals divided into subgroups on the basis of their age at admission (0–12, 13–36, and 37–60 hours) with healthy age-matched foals. The Mann-Whitney U-test was used to compare TH concentrations of surviving and nonsurviving foals at admission (t0), without any correction for age.
3. Results
Table 2 Means SD of plasma T3 and T4 concentrations (nmol/L) in foals affected by PAS (group 2) from admission to 7 days of hospitalization. TH
t0
t1
t2
t3
t4
t5
t6
t7
T3 T4
6.57a,b 4.67 259.79a 60.97
5.47b 2.27 188.87b 55.78
4.29b,c 2.02 162.89b 46.75
3.97b,c 1.83 141.85b 41.03
3.89a,c 1.57 108.42b 16.57
3.48a,c 1.16 101.54b 16.32
3.35a,c 1.22 71.49b 17.79
3.76a,c 1.01 58.78b 15.72
a,b,c
Different superscript letters in rows indicate significant differences between time points with GLM analysis (P <0.01).
A. Pirrone et al. / Theriogenology 80 (2013) 624–629
Statistical comparison between group 1 and group 2 foals divided into age groups showed significantly lower plasma T3 and T4 concentrations only in PAS foals (n ¼ 11) less than 12 hours old at admission (P < 0.01) (Table 3). 4. Discussion To the authors’ knowledge, this is the first study in which plasma TH concentrations (T3 and T4) were evaluated in neonatal foals affected by PAS, in comparison with a control group of clinically healthy foals. In healthy foals, this study showed a peak of plasma T3 concentration at 24 hours of age; it remained high for up to 48 hours and then showed a decreasing trend for the rest of the study period. These results are in agreement with the previous studies performed in neonatal foals [6,20], which showed plasma T3 concentrations seven times higher in neonates than in adults, with a progressive increase in the first 10 hours of life, peaking at 24 to 48 hours, and followed by a progressive decrease. However, this result disagrees with the data found by Panzani et al. [22], who reported a time-dependent decrease from birth in the first 2 weeks of life. On the other hand, plasma thyroxine concentration was high at birth in healthy foals and decreased significantly from as early as 24 hours of life. These data are partially in agreement with that reported by Panzani et al. [22], who found high T4 plasma levels at birth, followed by a significant decrease at 12 hours. Some studies reported plasma T4 concentrations at birth 14 times higher than in adults, with a subsequent rapid decrease to adult concentrations at 16 days of life [6,20]. A large standard deviation was found at t0, as previously reported by other authors [22,23]. Such a high TH concentration has been detected only in newborn foals, and they are higher than in any other species in any physiological stage. The high TH concentrations of newborn foals may be responsible for their high thermogenic capacity and remarkable rapidity of growth during the perinatal period, especially of musculo-skeletal and nervous systems [6]. Conversely, in human babies born at term, a further increase of both plasma T3 and T4 concentrations in the early hours of life has been reported [31]. Fisher et al. [32] hypothesized that this increase is due to a shift from a predominantly inactive thyroid gland to a state of hyperactivity, which allows an adequate adaptation of the newborn to the extrauterine environment. Moreover, the Table 3 Plasma TH concentration (nmol/L) in healthy foals (group 1) divided into three subgroups on the basis of age and in PAS foals (group 2) divided into three subgroups on the basis of age at admission. Foals
Number of samples
T3
Healthy (t0) PAS (0–12 h) Healthy (t1) PAS (13–36 h) Healthy (t2) PAS (37–60 h)
21 11 21 8 21 5
9.63 3.95 11.00 8.64 10.88 9.01
T4
4.76a 2.17b 2.76a 5.12a 2.53a 5.72a
394.81 242.64 249.5 288.97 210.85 250.86
140.8a 61.85b 82.73a 52.94a 47.20a 65.55a
a,b Different superscript letters in columns indicate significant differences with Mann-Whitney U-test (P < 0.01).
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increase in the concentrations of T4-binding globulins could be a further cause of total T4 increase [33]. In infants, the temperature variation between intrauterine and extrauterine environment appears to be able to stimulate the HPT axis, with an increased TRH and TSH secretion immediately after birth; this increase has a brief duration, with a peak at about 30 minutes after birth, followed by a progressive decrease in plasma TSH concentration at 3 to 5 days of life due to the T4 negative feedback [32]. In the present study, the monitoring of TH concentrations in foals affected by PAS showed a significant timedependent change from the fourth day of hospitalization in T3 concentrations and from 24 hours of hospitalization in T4 concentrations. It is worth noting the absence of the plasma T3 increase at t1 reported in healthy foals in the present study. The results of group 2 cannot be compared with those of other authors, because, to the authors’ knowledge, there is no study describing TH concentrations in foals affected by PAS. TH concentrations in sick foals were investigated by several authors. Silver et al. [24] evaluated T3 concentration in premature foals finding lower concentrations than in term foals. In a recent study of 26 sick foals with varying diagnoses (12 affected by PAS), Panzani et al. [22] reported lower plasma T3 concentrations than in healthy foals during the first week of life and lower plasma T4 concentrations during the first 3 days of life. In a study by Himler et al. [23], lower TH concentrations were observed in foals admitted to the ICU compared with healthy foals aged 12 to 36 hours, except for reverse T3. These authors reported that both the presence of disease and prematurity were associated with TH deficiencies, because of the lower values found in premature sick foals compared with term sick foals. The authors further suggested that the HPT axis immaturity of premature foals could aggravate the symptoms in animals debilitated by systemic illness. In human medicine, T4 concentration was lower in premature than in full-term babies due to the immaturity of the HPT axis, and the concentration was related to gestational age and birth weight [9]. Newborn human babies with respiratory distress syndrome had normal TSH, T3, T4, and fT4 concentrations at birth, and a significant decrease in T3, T4, and fT4 on the fifth day of life; TSH values remained rather normal [34]. The same data have been reported by Pereira and Procianoy [35] at 18 to 24 hours of life in infants undergoing a period of perinatal asphyxia. In another study, the same authors investigated the effects of perinatal asphyxia on THs concentrations, and they found statistically lower values in babies with moderate or severe hypoxic-ischemic encephalopathy compared with healthy ones [15]. In the present study, TH concentrations observed in PAS foals <12 hours old at admission were significantly lower than in healthy foals within 10 minutes from birth. This result is only partially comparable with those of other authors. Panzani et al. [22] found a similar result in premature, PAS, and septicemic foals compared with healthy foals, although TH concentrations were evaluated on the basis of foal’s age at each sampling time, regardless of the elapsed time from admission. A similar result was also obtained in sick foals (septic and nonseptic) by Himler et al.
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[23], who assessed TH concentrations only at admission independently of age and did not assess age-related changes in TH concentrations. As suggested by Himler et al. [23], in sick nonseptic and septic foals, the lower TH concentrations in foals affected by PAS found in this study could be the expression of nonthyroidal illness syndrome (NTIS or euthyroid sick syndrome). This is a well-recognized syndrome described in adult dogs and humans characterized by low TH concentrations in patients with severe nonthyroidal illness [36–40]. This syndrome is characterized by low T3 and elevated rT3 concentrations, associated with normal or low T4 and TSH concentrations [40]. NTIS probably represents an adaptive response to a systemic illness with a suppressive effect on the HPT axis and a decreased metabolism preventing organ dysfunction or death [36]. Initially, the conversion of T4 to T3 in peripheral tissues decreases, and, as the severity of illness, and often the associated starvation, progresses, T4 concentration also decreases, suggesting dysfunction at the hypothalamic, pituitary, or thyroid gland level [37]. As reported by Breuhaus [5], NTIS can also result in decreased TH-binding proteins and decreased binding protein affinity and capacity. This effect of illness decreases the concentrations of total T4 and T3, but has less effect on free fraction concentrations. Other causes of NTIS may be represented by circulating cytokines generated during inflammation or treatments with some medications such as glucocorticoids or amiodarone [41,42]. Van der Poll et al. [41] found that tumor necrosis factor is involved, either directly or indirectly, in the pathogenesis of this syndrome. Moreover, in healthy individuals injected with interferon-a, TSH and T3 decrease while reverse T3 increases. This biochemical picture is similar to euthyroid sick syndrome and may be mediated through interleukin-6 [42]. It is worth noting that hypoxia can also activate specific mediators, such as cytokines [26]. Aly et al. [43] found the concentrations of IL-1b, IL-6, and tumor necrosis factor-a in cerebrospinal fluid and serum significantly higher in human neonates with hypoxic-ischemic encephalopathy when compared with infants in the control group. Moreover, all three cytokines in the cerebrospinal fluid correlated significantly with the clinical severity of asphyxia. We suggest that cytokine increase during perinatal asphyxia could trigger the effects on the neonatal HPT axis causing the lower TH concentrations found in this study in PAS foals <12 hours old. Because assessment of NTIS was not a specific aim of the present study, plasma rT3 concentration was not evaluated; therefore, we do not have enough data to confirm that foals affected by PAS had NTIS. In the present study, a prognostic value of TH concentrations at admission was not found, probably due to the low number of nonsurviving foals (n ¼ 4); therefore, it warrants further investigations on a larger number of animals. A prognostic value of TH concentrations has been found by Himler et al. [23], with lower TH concentrations in nonsurviving premature and septic foals than in surviving premature and septic foals. Data obtained in this trial suggest, therefore, that PAS may cause lower T3 and T4 concentrations in affected foals than in age-matched healthy foals, as reported for other
systemic illnesses, such as sepsis and prematurity. Further studies are needed to find out if thyroid replacement therapy could be useful in the treatment of critically ill foals affected by PAS.
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