Allopregnanolone infusion induced neurobehavioural alterations in a neonatal foal: Is this a clue to the pathogenesis of neonatal maladjustment syndrome?
Reasons for performing the study: Increased plasma progestagen concentrations have been reported in foals with neonatal maladjustment syndrome (NMS). These steroids may cross the blood–brain barrier and have dampening effects in the central nervous system.
Objectives: To evaluate if the infusion of a progesterone derivative (allopregnanolone) in a healthy neonatal foal would induce clinical signs compatible with NMS.
Methods: A healthy neonatal foal from a healthy mare with a normal gestation (length, no complications), birth and placenta was infused with allopregnanolone to observe its neurobehavioural effects. Heparinised blood samples were collected pre- and post infusion to determine various progestagen concentrations using liquid chromatography mass spectrometry. A second healthy neonatal foal was infused with ethanol and saline for comparison of clinical observations.
Results: Infusion of allopregnanolone resulted in obtundation, lack of affinity for the mare and decreased response to external stimuli. These effects were short-lasting and associated with measurable concentrations of progestagens.
Conclusions and potential relevance: Infusion of a steroid metabolite to a healthy neonatal foal resulted in neurobehavioural alterations compatible with those observed in foals with NMS. These findings suggest that increased progestagen concentrations may be responsible for some of the behavioural changes observed in foals with NMS.
Neonatal maladjustment syndrome (NMS) is a common disorder of neonatal foals that manifests within the first 72 h of life (Bernard et al. 1995). The proposed mechanisms include hypoxic and ischaemic events prior to, during and shortly after parturition (Palmer and Rossdale 1976). Affected foals exhibit neurological dysfunction such as seizures and altered states of consciousness, behaviour and response to stimuli (Bernard et al. 1995; Ringger et al. 2011). However, hypoxic and ischaemic injury is not always identified upon histopathological evaluation, and long-term neurological deficits have been reportedly rare. Fetal corticosteroids, through activation of the hypothalamo-pituitary-adrenocortical (HPA) axis, contribute to the maturation of many organs and regulate the transition between intra- and extrauterine life (Rossdale 2004). Rossdale et al. (1995) reported increased concentrations of progestagens in neonatal foals that rapidly decrease over the following 48 h after birth (Houghton et al. 1991; Rossdale 2004). Foals with NMS have been reported to have persistently increased concentrations of plasma progestagens (Houghton et al. 1991; Rossdale et al. 1995; Rossdale 2004). Recently, concentrations of several plasma progestagens (progesterone, epitestosterone and androstenedione) were found to be significantly increased in foals with NMS compared with foals with other disorders (J.E. Madigan unpublished data). Progestagens called neurosteroids can cross the blood–brain barrier and have neuromodulatory effects (Mellon and Griffin 2002; Naert et al. 2007). It is proposed that in a subset of foals the signs of NMS may not be the result of hypoxia, and that these neurosteroids may play a role in the aetiology and clinical manifestations of foals with NMS. The aim of this study was to evaluate if the infusion of allopregnanolone in a healthy neonatal foal would induce clinical signs compatible with NMS.
Materials and methods
A healthy neonatal 50 kg Quarter Horse colt from the research herd at the School of Veterinary Medicine, University of California, Davis was selected for the infusion. The foal was born from a healthy Quarter Horse mare with a normal gestational length without complications, and normal observed parturition except that the placental passage was prolonged. Although retention of the placenta for 7 h post birth is not considered normal by most standards, the placenta was evaluated and determined to be complete and normal. Further, the mare did not display any signs or complications associated with placental retention. The foal was deemed healthy based upon complete physical and neurological examinations immediately post birth. The foal exhibited normal adaptive behaviour with righting reflex, and time to stand and suckle within normal limits. Repeat physical and neurological examinations at age 6 h, immediately prior to infusion, were also normal.
Intravenous catheters were placed aseptically in the right jugular vein for sample collection and in the left jugular vein for infusion of allopregnanolone. Allopregnanolone (5 alpha-pregnan-3 alpha-ol-20-one)a was dissolved in an ethanol-based solution to a total concentration of 9 mg/ml. Infused dose and concentration of allopregnanolone in this foal were determined based on concentrations reported in in vivo studies in the modulation of the HPA axis in male rats (Naert et al. 2007). An initial bolus of 0.05 mg/kg bwt i.v. of allopregnanolone was given followed by a constant rate infusion (CRI) of 0.02 mg/kg bwt/min using an infusion pump. Based on clinical effects of the initial dosage, a second bolus of 0.1 mg/kg bwt i.v. was given after 5 min and followed by a CRI of 0.04 mg/kg bwt/min for 5 min. The infusion was discontinued for 30 min to allow observation of any neurobehavioural (NB) alterations, and then a final bolus of 0.2 mg/kg bwt i.v. was given.
Neurobehavioural alterations were recorded and graded through a NB scoring system developed by the authors for the assessment of foals with NMS (Table 1). From preliminary work, foals with NMS had scores >8 from a range of 0 (normal foal) to 20 (comatose with paroxysmal activity) (JE Madigan, unpublished data). Mentation was defined as: normal if the foal was alert and responsive; quiet to obtunded if the foal was apparently lethargic but responsive to external stimuli (e.g. touch, sound); stuporous if level of consciousness was decreased but responsive to painful stimuli (e.g. pinching skin with haemostats); and comatose if the foal lost consciousness and was unresponsive to any stimuli. Paroxysmal activity was defined as abnormal events such as seizures or seizure-like activity, rhythmic limb movements, tremors or paddling. The NB scores were calculated at 5 min intervals throughout the infusion period. Following the infusion, the foal was observed hourly for the first 6 h and then at 12 h intervals for 2 days.
Table 1. Neurobehavioural scoring system (range 0–20)
|Mentation and reaction to stimuli||Normal, bright, alert responsive||Mildly obtunded, slightly decreased or increased reactivity to stimuli||Moderately obtunded, moderately decreased or increased reactivity to stimuli||Severely obtunded with hyper-reactivity to stimuli; if stuporous add +1 or if comatose add +2|
|Ability to stand||Stands unassisted||Stands with minimal assistance||Stands with marked support||Unable to stand|
|Bonding to mare||Actively bonds with and follows mare||Slightly reduced interaction with mare||Aimless wandering or periods of reduced responsiveness to mare||Unaware of mare|
|Ability to suckle||Latches on and suckles effectively||Searches out teat but does not suckle vigorously||Weak, ineffective suckling||No suckling|
|Paroxysms||None||0||None, limb stretching, paddling||Seizures or seizure-like activity|
|Ear position||Erect||0||Ears partially erect||Floppy, no tone|
Heparinised blood samples were collected at birth, age 6 h and at 15 min intervals during the infusion. Blood was immediately centrifuged following collection and plasma stored at -80°C until analysed by liquid chromatography mass spectrometry (LC-MS) utilising on-line 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 sulphate. Analytes were separated by liquid chromatography using a Thermo TLX-2 TFC system with a Thermo Cyclone P extraction column and an ACE C18 analytical column. Analytes were introduced by electrospray ionisation to a Thermo TSQ Vantage triple-quadrupole mass spectrometer operating in both negative and positive modes. Free 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 analysed: allopregnanolone, dehydroepiandrosterone (DHEA), 5-alpha dihydroprogesterone, 17-hydroxy pregnenolone, pregnenolone, pregnanediol and progesterone. A diagram of the relation of these neurosteroids is shown in Figure 1.
For NB comparison, a second age-matched clinically healthy neonatal Quarter Horse colt was infused with 99.9% ethanol diluted with 0.9% saline to a final concentration of 5% ethanol without allopregnanolone. Infusion of this solution followed the same protocol (dosage [based on 5% ethanol] and rate) of administration as for the first foal. The study was approved by an Animal Care and Use protocol from University of California, Davis.
Prior to the allopregnanolone infusion, the colt was bright, alert and responsive (NB score of 0; File S1). Infusion of 0.05 mg/kg bwt allopregnanolone followed by a CRI at 0.02 mg/kg bwt/min resulted in signs of sedation and decreased responsiveness to the environment (NB score of 14). Infusion of higher concentrations of allopregnanolone (0.1 and 0.2 mg/kg bwt) resulted in dramatic NB effects with the foal becoming recumbent, stuporous, unresponsive to the mare, environment, sound and tactile stimulation (NB score of 16; File S2–S4). Clinical signs persisted during the constant rate infusion. Within 8 min of cessation of the infusion the foal began to show signs of increased responsiveness. By 15 min after cessation of the infusion or the final bolus the foal was standing but continued to show clinical signs of mild obtundation, reduced coordination and poor udder seeking ability (NB score of 9; File S5). The foal appeared normal by 30 min after infusion of the neurosteroid (NB score of 0; File S6). No long-term NB effects were observed following the infusion. The control foal's NB scores were unchanged throughout the infusion. Steroid concentrations from Foal 1 are detailed in Table 2; these were not measured in the control foal due to lack of NB alterations, cost and probability of undetectable concentrations of neurosteroids based on preliminary work (JE Madigan, unpublished data). Due to an increase in dehydroepiandrosterone (DHEA) concentrations between birth and age 6 h, luteinising hormone (LH) concentrations at various time points were measured to investigate if this rise was due to production by the testis. However, there was no change in LH concentration.
Table 2. Steroid concentrations and NB score (0–20) in a healthy foal infused with allopregnanolone
|0||10 min post birth||0||NP||NP||37.9||1295.0||3.1||3074.3||ND||ND||110.7|
|6 ¼||Bolus 0.05 mg/kg bwt i.v., CRI 0.02 mg/kg bwt/min||14||2.1||58||74.5||74.5||0.8||1252.6||18.1||478.9||63.2|
|6 ½||15 min post initial infusion||9||2.6||55||1232.0||95.3||1.2||1206.7||18.8||126.6||73.5|
|6 ¾||Bolus 0.2 mg/kg bwt i.v.||16||4.7||40||56.9||57.6||0.6||1144.5||28.4||466.2||79.6|
Infusion of allopregnanolone to a healthy foal in this study produced marked NB effects. This is consistent with the clinical use of certain steroidal drugs, such as alphaxalone, as anaesthetic agents in male rats (Naert et al. 2007). Allopregnanolone in other species has been shown to cross the blood–brain barrier and is thought to mediate its effects in the central nervous system (CNS) via the GABAA receptor (Zhu et al. 2001). Infusion of allopregnanolone in this healthy foal provided evidence that 5-alpha reduced pregnanes can cross the blood–brain barrier and have effects in the CNS. Allopregnanolone concentrations peaked in conjunction with maximum NB effects following infusion. The rapid recovery from NB alterations with no apparent residual deficits once the infusion was discontinued, suggested that allopregnanolone was quickly metabolised in this healthy foal. Similar rapid dampening effects in the CNS and recovery were observed with the use of the neurosteroid anaesthetic alphaxalone in ponies undergoing castration (Leece et al. 2009). As allopregnanolone is apparently metabolised rapidly, the clinical signs associated with NMS in foals would be expected to dissipate rapidly. However, clinical manifestations of NMS can last several days, suggesting ongoing persistent production and release of allopregnanolone or other neurosteroids responsible for such observations. It is also unclear what triggers and stops these events in affected foals. Progestagen levels in this healthy foal decreased with age and are in agreement with the results of previous work (Holtan et al. 1991). The rise in DHEA between birth and age 6 h in this foal appeared to be neither testicular nor adrenal in origin as determined by constant levels of luteinising hormone and pregnanes, respectively, and was therefore deemed unlikely to be of biological relevance.
Recently, higher plasma concentrations of progesterone, epitestosterone and androstenedione were found in NMS foals compared with foals with other disorders (J.E. Madigan unpublished data). Findings from that work, along with the NB alterations induced by the infusion of allopregnanolone support our proposed hypothesis that NMS is in part a manifestation of persistent fetal HPA status mediated and sustained by elevated concentrations of progestagens as occurs naturally in the fetus (Warnes et al. 2004). The fetus must rapidly change from the quiet suppressed state in utero to one of arousal, and attempts to rise shortly after birth. A failure of the transition from the fetal HPA status to immediately post birth signals to engage the newborn into normal post foaling neurobehaviour may be the cause or involved in part in the pathogenesis of NMS. Further, the measured neurosteroids and altered neurological status in this study suggest that neurosteroids readily cross the blood–brain barrier and exert altering CNS effects compatible with NMS in affected foals. Certainly some foals suffer severe birth hypoxia and recover, and have been included in the broad description of NMS. However, the recovery from severe birth hypoxia would be expected to be slow and likely to have residual neurological deficits as documented in all other mammalian species suffering severe birth hypoxia (McAuliffe et al. 2006). We propose that ongoing production of pregnanes by the foal's brain and adrenal glands causes the clinical signs observed in foals with NMS and that rapid recovery of signs with no apparent residual deficits would be compatible with the decline of pregnane-mediated sedative type effects (Zhu et al. 2001).
It is unclear how foals that are normal at birth develop NMS within the first 48 h of life. However, we speculate that a similar mechanism reported in neonatal sheep may occur whereby neonatal stress can increase allopregnanolone production by the brain and release of deoxycorticosterone from the adrenal glands, which the brain metabolises into 5α-tetrahydrodeoxycorticosterone (TH-DOC), another CNS depressant (Hirst et al. 2008). Obtundation, seizures and hyperaesthesia are common signs of NMS. Whilst the infused neuroactive steroid allopregnanolone has a dampening effect in the CNS, others within the large spectrum of neurosteroids, including metabolites of allopregnanolone, have excitatory effects that may be associated with seizures and hyperaesthesia (Rogawski and Reddy 2004). Neurosteroid concentrations in clinical NMS are likely to be a far more complex condition than that represented by infusion of one compound. Further work is indicated to evaluate the role of pregnanes in foals with NMS.
Authors' declaration of interests
No conflicts of interest have been declared.
Source of funding
Funding was provided by the Center for Equine Health with funds provided by the State of California pari-mutuel fund, and contributions by private donors to the equine neurological study group at UC Davis.
The authors thank Dr Janet F. Roser from the Department of Animal Science, University of California, Davis, California.
a Steraloids Inc., Newport, Rhode Island, USA.