Analytical Clinical Studies
Abnormal plasma neuroactive progestagen derivatives in ill, neonatal foals presented to the neonatal intensive care unit
M. ALEMAN*, K. J. PICKLES, A. J. CONLEY†, S. STANLEY‡, E. HAGGETT§, B. TOTH# and J. E. MADIGAN
Department of Medicine and Epidemiology, School of VeterinaryMedicine University of California, USA †Department of Population Health and Reproduction, School of VeterinaryMedicine University of California, USA ‡California Animal Health & Food Safety Laboratory System (CAHFS), School of VeterinaryMedicine University of California, USA §Rossdales Equine Hospital, Suffolk, UK #Department of Veterinary Clinical Sciences, Purdue University, Indiana, USA. *Correspondence email: firstname.lastname@example.org; Received: 16.03.11; Accepted: 18.01.13
Reasons for performing the study: Increased levels of pregnanes have been reported in foals with neonatal maladjustment syndrome (NMS). These steroidsmay cross theblood–brain barrier andhavedepressive 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 andpotential 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 pregnenolonemeasurementmay be useful in aiding diagnosis of NMS.
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Keywords: horse; foals; hypoxia; neurosteroids; pregnanes; progestagens
CNS: central nervous system
LC-MS: liquid chromatographymass spectrometry
NMS: neonatal maladjustment syndrome
SIM: single ionmonitoring
SRM: select reactionmonitoring
TFC: turbulent flow chromatography
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 . Histopathological evidence of cerebral haemorrhage and hypoxia has been detected in some severely affected foals . However, many foals do not have histological evidence of hypoxia, oedema or haemorrhage . 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 oftenmanifest [8–10]. The fast recovery with no apparent longterm 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 . Increased concentrations of plasma pregnanes and a correlation between decreasing levels of pregnanes and clinical recovery have been previously reported .
Certain steroidal compounds, predominantly 5a-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  and are potent allosteric modulators of the GABAA receptor; low concentrations cause weak enhancement of GABA activity and high concentrations cause complete noncompetitive inhibition .
Infusion of certain 5a-reduced pregnanes into rats and mice [16,17] and neonatal foals  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. bs_bs_banner
Equine Veterinary Journal ISSN 0425-1644
DOI: 10.1111/evj.12065 661Equine Veterinary Journal 45 (2013) 661–665© 2013 EVJ Ltd
Materials and methods
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) . Foals with a sepsis score of 11 or greater were additionally classed as septic . 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.