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Keywords:

  • horse;
  • foals;
  • hypoxia;
  • neurosteroids;
  • pregnanes;
  • progestagens

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Authorship
  11. References
  12. Supporting Information

Reasons for performing the study

Increased levels of pregnanes have been reported in foals with neonatal maladjustment syndrome (NMS). These steroids may cross the blood–brain barrier and have depressive effects in the central nervous system leading to behavioural abnormalities and altered states of consciousness in affected foals.

Objectives

The aim of this study was to determine the pregnane profile of foals with NMS and compare it with that of healthy controls and sick, non-NMS foals.

Study design

Prospective-clinical study.

Methods

Thirty-two foals with a clinical diagnosis of NMS, 12 foals with other neonatal disorders and 10 healthy control foals were selected for the study. Heparinised blood samples were collected from each group of foals and pregnane and androgen concentrations determined using liquid chromatography mass spectrometry at 0, 24 and 48 h of age.

Results

Healthy foals showed a significant decrease in pregnane concentrations over the first 48 h of life (P<0.01). Foals with NMS and sick, non-NMS foals had significantly increased progesterone, pregnenolone, androstenedione, dehydroepiandrosterone and epitestosterone concentrations compared with healthy foals (P<0.05). Progesterone and pregnenolone concentrations of sick, non-NMS foals decreased significantly over 48 h (P<0.05), whereas concentrations in NMS foals remained increased.

Conclusions and potential relevance

Pregnane concentrations of ill, neonatal foals remain increased following birth, reflecting a delayed, or interrupted, transition from intra- to extra-uterine life. Serial progesterone and pregnenolone measurement may be useful in aiding diagnosis of NMS.


Abbreviations
CNS

central nervous system

DHEA

dehydroepiandrosterone

HPA

hypothalamic-pituitary-adrenocortical

LC-MS

liquid chromatography mass spectrometry

NMS

neonatal maladjustment syndrome

SIM

single ion monitoring

SRM

select reaction monitoring

TFC

turbulent flow chromatography

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Authorship
  11. References
  12. Supporting Information

Neonatal maladjustment syndrome (NMS) is one of the most common diseases affecting foals within the first 72 h of life [1, 2]. The disorder has been referred to as hypoxic–ischaemic encephalopathy, perinatal asphyxia, neonatal encephalopathy and dummy foal syndrome [3, 4]. The proposed pathogenesis is the result of hypoxia and ischaemia of the brain that occurs shortly before, during or after parturition leading to neuronal cellular energy failure and death [3, 5, 6]. Clinical signs are consistent with brain hypoxia and include alterations in the state of consciousness, abnormal behaviour and paroxysmal activity such as paddling and seizures [7]. Histopathological evidence of cerebral haemorrhage and hypoxia has been detected in some severely affected foals [7]. However, many foals do not have histological evidence of hypoxia, oedema or haemorrhage [1]. Furthermore, many foals have a normal birth and recover quickly and fully from the condition. This is in contrast to asphyxiated infants and animal models of perinatal asphyxia in which a significantly longer recovery time is needed and long-term neurological deficits are often manifest [8-10]. The fast recovery with no apparent long-term deficits and lack of evidence of hypoxia or ischaemia in affected neonatal foals suggest that the syndrome may not be exclusively the result of hypoxia.

Neonatal foals have high concentrations of pregnanes at birth that decrease rapidly over the first 48 h of life [11]. Increased concentrations of plasma pregnanes and a correlation between decreasing levels of pregnanes and clinical recovery have been previously reported [12]. Certain steroidal compounds, predominantly 5α-reduced pregnanes, appear to have important neuromodulatory roles [13-15]. These steroids are synthesised de novo in glial cells from cholesterol or blood-borne steroid precursors [15] and are potent allosteric modulators of the GABAA receptor; low concentrations cause weak enhancement of GABA activity and high concentrations cause complete noncompetitive inhibition [13]. Infusion of certain 5α-reduced pregnanes into rats and mice [16, 17] and neonatal foals [18] leads to anaesthesia or marked behavioural effects suggesting that these pregnanes cross the blood–brain barrier and exert neuromodulatory effects.

We propose that NMS may comprise of more than one phenotype: foals with hypoxia and ischaemia and foals with persistence of fetal hypothalamic-pituitary-adrenocortical (HPA) axis and increased pregnanes (pregnenolone, progesterone and metabolites) that recover rapidly with no apparent residual neurological deficits. The aim of this study was to determine the steroid profile of foals with NMS and compare it with that of foals with other neonatal diseases and healthy control foals.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Authorship
  11. References
  12. Supporting Information

Foals

The NMS foal group (n = 32; 15 colts and 17 fillies) and the other neonatal disease foal group (n = 12; 4 colts and 8 fillies) comprised foals admitted to the University of California, Davis Veterinary Medical Teaching Hospital in 2008 and Rossdale and Partners, Newmarket, UK in 2010 and 2011. To be included as a foal with NMS other disorders with a similar clinical presentation, such as prematurity and sepsis, were ruled out based on a minimum database (published sepsis score, complete blood count, chemistry panel, blood gases, indirect blood pressure, central venous pressure, blood culture, urinalysis, abdominal ultrasound and carpi, tarsi, thoracic and abdominal radiography) [19]. Foals with a sepsis score of 11 or greater were additionally classed as septic [19]. Historical knowledge of pre-, intra- or post natal hypoxia was recorded. Clinical signs of NMS included altered mentation (obtunded, stuporous, comatose), decreased bonding to the mare, vocalisation, aimless wandering, hyper- or lack of reactivity to stimuli, seizures and abnormal ear position. Foals were subjectively scored by the attending clinician as mild-moderate if able to nurse and ambulate with help or severe if recumbent and unable to nurse, even with help. Case details of NMS foals are given in Table S1. Foals in the other neonatal disease group (sick, non-NMS controls) were randomly selected based on client consent and availability of the authors for sample collection. These foals had a variety of clinical diagnoses (Table S2).

A third group of healthy control neonatal foals (n = 10; 4 colts and 6 fillies) was recruited from the 2009 and 2010 foal crops at the Center for Equine Health, University of California, Davis. Inclusion criteria for control foals included a term birth (>320 days gestation) with normal, uncomplicated delivery and physical examination.

All foals were less than 48 h of age at enrolment into the study. No attempts were made to standardise treatments given to the foals during hospitalisation. Outcome was recorded as survival to discharge. The study was approved by the University of California Institutional Animal Care and Use Committee and client consent obtained prior to enrolment in the study.

Sample collection and analysis

Heparinised blood was collected from healthy control foals at 0, 24 and 48 h following birth. Samples were collected from NMS foals and other neonatal disease foals after initial stabilisation and thereafter at the designated 24 and 48 h time points as appropriate. For foals presenting at birth, samples were collected within 2 h of parturition.

Whole blood was immediately centrifuged after collection and plasma stored at -80°C until analysed. Plasma was analysed by liquid chromatography mass spectrometry (LC-MS) utilising online sample extraction by turbulent flow chromatography (TFC) and detection by select reaction monitoring (SRM) on a triple quadrupole mass spectrometer. Samples were diluted 2:1 with water fortified with 4 internal standards: D3-Testosterone, D3-Boldenone, D7-Androstenedione and D3-Testosterone Sulfate. Analytes were separated by liquid chromatography using a Thermo TLX-2 TFC systema with a Thermo Cyclone P extraction columna and an ACE C18 analytical columnb. Analytes were introduced by electrospray ionisation to a Thermo TSQ Vantage triple-quadrupole mass spectrometerc operating in both negative and positive modes. Free steroid concentrations of 34 steroids were monitored in one analytical method over a 24 min run time. Detection and quantitation was accomplished using 3 or more SRM transitions per compound for all compounds other than 17-hydroxy pregnenolone where single ion monitoring (SIM) was utilised. This method was validated and the following assessed for each analyte: linearity, limit of detection, limit of quantitation, accuracy, precision, matrix effects, extraction recovery and potential endogenous interferences. The following steroids were evaluated: pregnanes including progesterone, 17-hydroxy-progesterone, 5α-dihydroprogesterone, pregenolone, allopregnanolone and pregnanediol; androgens and oestrogens including nandrolone sulfate, boldenone sulfate, 17-β oestradiol sulfate, testosterone sulfate, 1,4-androstadiene-3,17-one, testosterone glucoronide, 19-norandrostenedione, boldenone, androstenedione, nandrolone, oestrone, testosterone, epinandrolone, epitestosterone, 6-α-hydroxyandrostenedione, nandrolone glucuronide, 17-β oestradiol, 17-α oestradiol, 19-norepiandrosterone, dehydroepiandrosterone (DHEA), DHEA-sulphate, 17-hydroxypregnenolone, 5-α dihydronandrolone, 5-α-estran-3-β-17-α diol, 5-α dihydrotestosterone, 19-nor-androsterone, 5-β dihydrotestosterone, oestrone sulfate. These steroids were chosen due to convenience of a pre-existing, extensive steroid panel.

Data analysis

Descriptive data are reported as median and ranges. Friedman tests were used for repeated measures analysis of steroid concentrations of healthy foals. Kruskal–Wallis tests were used for multiple group comparisons. Following a significant Kruskal–Wallis test, Mann–Whitney tests were used for nonpaired 2 group comparisons with Bonferroni–Holm correction. Nonparametric tests were chosen based on the failure of the data to conform to normal distributions using a Kolmogorov and Smirnov test and inability to transform the data using conventional methods. Level of significance was set at P<0.05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Authorship
  11. References
  12. Supporting Information

On presentation, 19 NMS foals were graded as mild-moderate and 13 as severe. Altered states of consciousness of foals with NMS ranged from mildly obtunded to stuporous to comatose.

Several progestagens were detected in extremely low concentrations (data not shown). Five steroids (both pregnanes and androgens) were consistently identified among foal samples: progesterone, pregnenolone, androstenedione, DHEA and epitestosterone. Healthy foals showed progressive, significant decreases in these steroids over the first 48 h of life (progesterone P<0.0001; pregnenolone P<0.0001; androstenedione P = 0.009; DHEA P = 0.006; epitestosterone P = 0.004) (Fig 1). There was no significant difference in healthy foal pregnane or androgen profiles between genders.

figure

Figure 1. Median and interquartile range plasma steroid concentrations (ng/ml) of a) androstenedione, b) dehydroepiandrosterone (DHEA), c) epitestosterone, d) progesterone and e) pregnenolone for healthy, sick control and neonatal maladjustment syndrome (NMS) foals during the first 48 h of life.

Download figure to PowerPoint

Compared with healthy foals, NMS foals showed increased concentrations of androstenedione (P = 0.02) and progesterone (P = 0.04) at 0 h (within 2 h of birth), androstenedione (P = 0.0002), DHEA (P = 0.001), epitestosterone (P = 0.0004), progesterone (P = 0.0001) and pregnenolone (P = 0.0007) at 24 h of age and androstenedione (P = 0.0008), DHEA (P = 0.007), progesterone (P = 0.0001) and pregnenolone (P = 0.003) at 48 h of age (Fig 1, Table 1). Sick control foals also had significantly increased concentrations of progesterone (P = 0.001) at 0 h, androstenedione (P = 0.005), DHEA (P = 0.003), progesterone (P = 0.01) and pregnenolone (P = 0.0009) at 24 h and androstenedione (P = 0.004), progesterone (P = 0.0004) and pregnenolone (P = 0.0006) at 48 h compared with healthy foals (Fig 1, Table 1). Compared with sick control foals, NMS foals had significantly higher concentrations of epitestosterone at 0 and 24 h of age (P = 0.02, P = 0.002, respectively). In contrast, sick control foals had significantly higher progesterone concentrations than NMS foals at 0 h (P = 0.01). While pregnane concentrations of sick control foals remained increased above those of healthy foals, their progesterone and pregnenolone concentrations decreased significantly (P = 0.02, P = 0.04, respectively) over 48 h. In contrast, steroid concentrations of NMS foals remained increased and showed a trend of increasing concentration over time (Fig 1).

Table 1. Median (range) serum steroid concentrations (ng/ml) in healthy, sick control and neonatal maladjustment syndrome (NMS) foals at 48 h of age
Steroid (ng/ml)Healthy controls (n = 10)Sick controls (n = 12)NMS (n = 32)P value (Kruskal–Wallis)
  1. DHEA = dehydroepiandrosterone; nd = not detectable. Groups with differing superscripts are significantly different (Mann–Whitney).

Androstenedionenda (nd–0.51)1.15b (nd–7.65)6.56b (nd–57.34)<0.0001
DHEA7.68a (nd–117.90)101.14a,b (nd–1412.60)92.98b (nd–1 511.06)0.028
Epitestosteronend (nd–0.15)nd (nd–13.20)0.53 (nd–11.53)0.1
Progesteronenda (nd–0.09)6.09b (1.97–14.28)14.22b (0.75–73.61)<0.0001
Pregnenolone103.76a (5.6–313.20)1119.40b (248.26–3416.08)1922.08b (nd–15,917.33)0.001

When considering the NMS foal population, foals with NMS alone had significantly higher DHEA concentrations than foals with NMS and another disease (P = 0.02). There was no significant difference in pregnane concentrations between mild-moderate and severely affected NMS foals (data not shown). When considering all ill foals admitted to the neonatal intensive care unit (NICU) (i.e. collating NMS and sick control foal data), there was no significant difference in pregnane concentrations between survivors and nonsurvivors, septic and nonseptic individuals and foals with and without a known history of hypoxia (data not shown).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Authorship
  11. References
  12. Supporting Information

The results of this study confirm that there are differences in the pregnane profiles of neonatal healthy foals, foals with NMS and foals with other clinical diagnoses. Pregnane concentrations of healthy neonatal foals declined rapidly, to essentially zero, within 48 h of birth in agreement with the study by Houghton et al. [11]. The fetal foal is subjected to high levels of progesterone and other progestagens in utero [20], deemed important in providing tonic inhibition of fetal central nervous system (CNS) activity and damping movement to prevent maternal damage [21]. Injections of progesterone or its metabolites into the ovine fetal circulation in late gestation reduce fetal electroencephalograph, electrocorticograph and electrooculograph activity, breathing movements and behavioural arousal, while inhibition of placental progesterone enhance these parameters [22-25]. The loss of placentally-derived precursors at birth and the switch to adrenal or other derived precursors causes this dramatic decline in pregnane concentrations shortly after birth in healthy neonates [26].

Apart from epitestosterone concentrations of sick control foals, foals presenting ill to the NICU (i.e. NMS and sick control foals) had higher concentrations of all measurable pregnanes than healthy controls within 2 h of birth. Pregnane concentrations of NMS foals remained increased over the 48 h time period in contrast to those of sick control foals that had significantly lower progesterone and pregnenolone concentrations at 48 h compared with birth. Serial blood sampling with continued elevation or increasing pregnane concentrations over 48 h of age may therefore prove useful in aiding diagnosis and possibly prognosis of NMS; however, further work is required to validate this possibility. These observations support the hypothesis of a delayed, or interrupted, conversion from intra- to extra-uterine life in ill, neonatal foals, particularly those with NMS. This mechanism may be similar to that reported in foals of mares treated with the progestagen altrenogest, which have a slower adaptation to the extra-uterine environment [27]. These steroids are suspected to be of adrenal origin based on extensive studies of neonatal lamb neurosteroid production [21]. Neonatal foals in this study and that of Rossdale [28] showed endogenous rises in neurosteroid concentrations thus eliminating placental origin.

The higher DHEA concentrations in NMS foals compared with foals diagnosed with NMS and another disease suggests different adrenocortical responses in these foal subsets. Pregnane profiles did not appear to differ between mild-moderate and severely affected foals although it is likely that a larger population needs to be sampled to detect such differences. Furthermore, the categorisation used may have been inappropriate for finding such differences. In previous studies, pregnane concentrations decreased in foals with NMS as they displayed clinical improvement [12]. This observation could not be validated by the current study as concentrations were only measured over the first 48 h of life. Pregnane concentrations were not significantly different between survivors and nonsurvivors in this study and, again, the short sampling period is likely to have precluded the ability to detect this finding. The effect of a known hypoxic episode on plasma pregnane profiles was examined due to the suggested aetiological role of hypoxia in NMS. Hypoxia did not appear to have an effect on pregnane concentrations; however, it is impossible to accurately evaluate this criterion, particularly with regard intra-uterine hypoxia.

Differences in concentrations of pregnenolone and pregnanediol between sick and healthy foals have previously been described [12]. Pregnanediol was not consistently measurable in the current foal population whereas the androgens, androstenedione and epitestosterone have not previously been identified in foals with NMS [29]. It is likely that these analytes were simply not investigated in the analytical method originally developed [11]. The use of LC-MS allows better differentiation of the individual steroids than can be achieved by radioimmunoassay [28].

The cause of the increased plasma pregnane concentrations detected in ill neonatal foals cannot be elucidated from this study; however, the authors propose that these concentrations occur as a result of persistence of fetal signals for the in utero state of being quiet and nonambulatory. Certain pregnanes, such as progesterone and their metabolites have neuromodulatory, anaesthetic and anxiolytic properties important for tonic inhibition of fetal CNS activity and damping fetal movement to prevent maternal damage [21]. The receptors in the fetal brain are more sensitive to these pregnanes, compared with the receptors in the adult brain [30]. Infusion of the neurosteroid pregnane allopregnanolone to a healthy neonatal foal induced obtundation, lack of affinity for the mare and decreased response to external stimuli [18]. These effects were short-lasting and associated with measurable concentrations of pregnanes [18]. This suggests that these steroids can cross the blood–brain barrier and exert neuromodulatory effects, which at high concentrations may have a dampening effect in the CNS with resulting alterations in states of consciousness, altered behaviour and responsiveness to stimuli, such as observed in NMS cases. Specific enzymes may be inhibited in these foals and the roles of 5α-reductase, 3β-hydroxysteroid dehydrogenase and 3α-hydroxysteroid dehydrogenase need to be further evaluated. It has been suggested that the 5α-reduction step may be critical in determining the quantity of 5α-reduced pregnane metabolites either produced from progesterone within the fetal brain or derived from precursors entering the brain from the blood [31]. The underlying cause of any possible abnormal adrenal function is also not known; it may reflect a state of dysmaturity in which the foal fails to transition to extra-uterine life or may reflect hypoxic injury to the HPA axis [28]. Another potential reason for persistence of fetal hormones is a failure of normal events of parturition that are essential for the transition from the in utero fetal cortical status to extra-uterine behavioural status. Regulation of the neuroactive steroid content in the fetal ovine brain is independent of adrenal steroidogenesis and hypothalamic–pituitary factors [32]; however, in the neonate, concentrations of some neurosteroids and their precursors in the peripheral circulation dramatically affect concentrations in the brain [31]. Lastly, another possible mechanism would be the reversion to fetal cortical status when adverse post birth circumstances occur. The syndrome of reversion to fetal circulation is a well known and accepted consequence of adverse birth and post birth events, which is seen in both maladjusted and foals with other neonatal diseases and causes the neonate to revert to mechanisms that regulated the cardiovascular system in utero.

It is also possible that the increased pregnanes are acting in a neuroprotective role as has been reported in other species. Stress (hypoxia, endotoxin) in the neonatal period increases neurosteroid concentrations in the brain of newborn lambs [26, 33], suggested to represent an endogenous protective mechanism. Similarly, acute, but not chronic, hypoxic stress during pregnancy increases fetal neurosteroid concentrations [26]. Indeed, inhibition of neurosteroid synthesis increases asphyxia-induced brain injury in late gestation fetal sheep [34].

Phenotypical characteristics of ‘maladjusted foals’ may have more than one aetiology (hypoxic/ischaemic vs. nonhypoxic/ischaemic). We speculate that the nonhypoxic foal is the one that lacks the normal transition from synthesis to inhibition of specific neurosteroids for readiness for birth (from fetal to neonatal neurosteroid profile). This may explain why some affected foals have a relatively fast recovery with no remaining long-term neurological deficits and no apparent or known hypoxic events prior, during or shortly after birth.

Plasma concentrations of progestagens were measured in this study but ideally concentrations in brain tissue, known to be much higher than those in the peripheral circulation, would be measured. Neurosteroids and their precursors are known to cross the blood–brain barrier [35] and are extremely potent such that small concentrations can have large local effects in neuronal tissue. Further, many steroids are metabolised to other compounds prior to exerting their effects.

In conclusion, specific alterations in pregnane profiles were detected between healthy control foals and ill, neonatal foals presenting to NICU. The anaesthetic and sedative properties of these pregnanes may account for the behavioural alterations seen in maladjusted and ill foals. These differences may reflect a delayed or interrupted transition from fetal to neonatal HPA status. Repeated measurements of these pregnanes over time may be useful for distinguishing between foals with NMS and other neonatal disorders. Increased pregnane concentrations may cross the blood–brain barrier and be responsible for some of the behavioural and neurological alterations observed in foals with NMS.

Authorship

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Authorship
  11. References
  12. Supporting Information

M.A. and J.E.M. were responsible for concept, design, study execution, data collection and interpretation and preparation of the manuscript. K.J.P. was responsible for design, data collection, study execution, data analysis and interpretation and preparation of the manuscript. A.J.C. and S.S. were responsible for data analysis and interpretation and preparation of the manuscript. E.H. was responsible for data collection, study execution, data interpretation and preparation of the manuscript. B.T. was responsible for data collection, study execution and preparation of the manuscript.

Manufacturers' addresses
  1. aThermo Scientific Inc., Franklin, Massachusetts, USA.

  2. bMAC-MOD, Chadds Fort, Pennsylvania, USA.

  3. cThermo Scientific Inc., San Jose, California, USA.

References

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Authorship
  11. References
  12. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Authorship
  11. References
  12. Supporting Information
FilenameFormatSizeDescription
evj12065-sup-0001-chinese.docx14K

Summary in Chinese.

evj12065-sup-0002-item1.doc63K

Table S1: Case histories of neonatal maladjustment syndrome (NMS) foal group (n = 32). Age = age at presentation; M = male; F = female; E = euthanasia; S = survival; Sev = severe NMS; M-M = mild-moderate NMS; PMN = polymorphonuclear cell count.

evj12065-sup-0003-item2.doc43K

Table S2: Case histories of sick control foal group (n = 12). Age = age at presentation; M = male; F = female; E = euthanasia; S = survival; ARDS = acute respiratory distress syndrome; PMN = polymorphonuclear cell count; n/a = not available.

evj12065-sup-0004-si.mp313376K

EVJ Podcast, No. 2, October 2014 - BEVA 2014 Scientific Review Internal Medicine

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