Since the 1970s, corticosteroids have been used with great success to reduce mortality and morbidity in preterm infants. 1 Randomized trials found no neurologic or cognitive effects in children who were treated prenatally with single courses of corticosteroids and followed up between 3, 6, and 12 years of age. 2–4 Administrating a single course of corticosteroids is now standard practice. In 1995, the US National Institutes of Health recommended that all infants at risk of preterm delivery receive single courses of corticosteroids to improve the survival rates. 5
There has been a trend to increase the number of treatments given to pregnant women at risk of preterm delivery, particularly in situations when risk persists or recurs after initial courses. A recent survey of obstetric practice in Australia found that 85% of clinicians would use repeated injections of corticosteroids in such cases, and half would do so weekly. 6 That practice has arisen without safety data; however, a randomized, controlled trial in humans on the effects of repeated courses of corticosteroids has recently started in Australia at multiple centers and is coordinated by the University of Adelaide (Australian Collaborative Trial of Repeated Doses of Steroids; personal communication, Dr. Caroline Crowther). Other trials in the United States and the United Kingdom are under development.
A number of animal studies documented detrimental effects of repeated corticosteroid doses on short- and long-term brain development. 7–11 Many of those studies were considered of questionable clinical relevance 5 because they used small laboratory mammals (rats, mice, and rabbits) in which corticosteroid treatment occupied a large portion of their short gestation and rapid postnatal development periods. Variations in route of administration—namely, maternal or direct injection into fetuses or newborn pups—are now known to alter outcome. 12
Previous data from our laboratory indicated that repeated corticosteroids delay myelination of the ovine optic nerve, 13 urgently suggesting that we extend our observations to other white-matter tracts. We compared the effects of clinically appropriate single- and repeated-corticosteroid doses on brain growth in sheep, chosen because of their extensive use as experimental models for human pregnancy. 14 The sheep brain is relatively mature at birth, similar to the human brain, 15 and thus is appropriate for examining effects of corticosteroids on brain growth. We chose maternal injection because it is currently standard clinical practice.
Materials and Methods
The effects of corticosteroids on organ weights were reported on animals in this study. 16 Ewes were datemated, then singleton pregnancy was confirmed in each case by ultrasound examination at 85 days' gestation. All ewes were injected intramuscularly at 100 days' gestation with 150 mg medroxyprogesterone acetate (Depo Provera; Upjohn, Rydalmere, Australia) to minimize risks of preterm births.
Pregnant ewes were ear-tagged at mating; then, a random number sequence was used to assign them to one of the three experimental groups and to preterm (day 125) or term (day 145) delivery. In the single corticosteroid group (n = 12), ewes received an intramuscular injection of betamethasone (Celestone Chronodose, 0.5 mg/kg; Schering Plough, Baulkham Hill, Australia) on day 104 of gestation followed by three injections of an equivalent volume of sterile normal saline on days 111, 118, and 124 of gestation. In the repeated corticosteroid group (n = 12), betamethasone was given intramuscularly at all four time points. Control ewes (n = 12) received intramuscularly an equivalent volume of sterile normal saline at the same time points as the single- and repeated-corticosteroid groups. All injections were carried out by a single operator (JAQ) and the group allocation was masked to the other investigators.
Cesarean delivery was done on pregnant ewes after premedication with intramuscular ketamine (12 mL, 100 mg/mL) and spinal anesthesia (4 mL, 2% lidocaine). Newborn lambs were weighed before terminal anesthesia with intravenous ketamine (1 to 2 mL, 100 mg/mL) and perfusion by ascending aorta with saline, followed by Karnovsky's fixative in cacodylate buffer (0.1 mol/L 300 mosm, pH 7.4).
Fetal brains were removed and whole-brain weights and volumes measured. The maximum cerebral anterior-posterior length, width, and depth were measured with vernier callipers. The cerebellum with brain stem attached was removed as one piece by cutting through the base of the inferior colliculus. The two structures were then separated and weighed individually. The weights and volumes of the cerebra were measured.
The sample of six animals per group was chosen to determine, with 80% power, a difference of 6 g in brain weight, assuming the standard deviation for the control animals to be 3 g, adjusting the type 1 error rate for multiple comparisons. Analysis of variance was used to compare each of the control and single- and repeated-corticosteroid groups. Maternal body weight, lamb gender, and gestational age were included in the analysis as covariates because they also affect the outcome. The Tukey-Kramer multiple-comparison adjustment was used to calculate the significance levels. P < .05 was considered statistically significant.
The project was approved by the Animal Ethics and Experimentation Committee of The University of Western Australia and compiled with the Australian National Health and Medical Research Council's Code of Practice for the Use and Care of Animals for Experimentation.
Table 1 provides the data of maternal body weights, lamb birth weights, and gestational ages. As we reported, lamb weights were significantly reduced. 16 There were no significant differences in baseline variables of maternal weight or gestational age. Tables 2 and 3 summarize brain growth data for the preterm and term animals in the control and single- and repeated-corticosteroid groups, respectively.
In the single corticosteroid group, after delivery at 125 days' preterm gestation, whole-brain weights were not significantly different compared with controls (Table 2). There were no significant differences between the single-corticosteroid group and controls in whole-brain volume, cerebral weight and volume, cerebellar and brain-stem weights, and maximum cerebral depth. Maximum cerebral anterior-posterior lengths and depths were significantly reduced compared with controls.
At term, the single-corticosteroid group showed significant reductions compared with controls in whole-brain weight and volume, cerebral volume, cerebellar weight, and maximum cerebral width and depth (Table 2). The remaining measures, cerebral and brain-stem weights and maximum cerebral anterior-posterior lengths, showed no significant differences.
Repeated corticosteroids had more profound effects on brain growth than single doses. After preterm delivery, the repeated-corticosteroid group showed significant reductions in weights and volumes of whole brains and cerebra, and maximum cerebral anterior-posterior lengths, widths, and depths (Table 3). The cerebellar and brain-stem weights did not show significant reductions. Figures 1 and 2 (upper rows) present examples of brains and cerebella, respectively, from preterm control, and single- and repeated-corticosteroid animals, showing the smaller sizes in the repeated-corticosteroid group.
At term, the repeated-corticosteroid treated group showed significant reductions in all measurements (Table 3). Figures 1 and 2 (bottom rows) show representative examples of brains and cerebella in the term control and single- and repeated-corticosteroid animals showing smaller sizes and disrupted vermal cerebellar surfaces resulting from corticosteroid treatment.
Single and repeated doses of corticosteroids retarded brain growth in fetal sheep; repeated doses had more profound effects, particularly at term.
The clinical dose of betamethasone (Table 4) 17–21 results in a 75% occupancy of corticosteroid receptors in the lung; higher or more frequent doses have little additional benefit. 5 The dosage for the current study was similar to that used clinically (Table 4) and conformed to previous studies in sheep in which fetal lung maturation was shown. 21 Measurement of fetal plasma betamethasone concentrations in humans and sheep found similar courses in peak values and rates of clearance (Table 4). Corticosteroids resulted in adrenal suppression in humans and sheep (Table 4). Humans and sheep respond similarly to corticosteroids in terms of lung maturation and adrenal suppression; the challenge is to find similarities in neurodevelopmental outcome.
There is much evidence from small laboratory mammals that shows exogenous corticosteroids retard brain development with a concomitant mosaic of biochemical, 7 structural, 8 and behavioral 22 deficits. Small laboratory mammals are considered inappropriate for examining outcomes of repeated-corticosteroid administration in a clinical setting, but their use has indicated a fundamental retardation of brain growth. For example, prenatal treatment administered maternally during the last week of pregnancy in rats consistently decreased brain weights in animals at preterm or term, 23 or within the first 2 to 4 weeks of life. 24 Examination at approximately 2 months postnatally found a catch-up in brain weight after a characteristic initial delay. 11 Treatment by direct injection into mouse and rat pups between 2 and 7 days postnatally resulted in reduced brain weights at 1 month 7 and at over 1 year. 22
There are few studies in larger, more clinically relevant experimental animals on effects of maternally administered exogenous corticosteroids on brain growth. Exposure of rhesus monkey fetuses to corticosteroids on days 132 and 133, and examination by day 135 (term = 165) found a trend towards lower brain weights, although reductions were not significant. 10 Hippocampal pyramidal cell numbers were reduced and neuronal and synaptic damage was evident. The interval between administration and delivery was too short to expect significant brain-weight reductions. Significant reductions in brain weight were found in rhesus monkeys after daily maternal injection between days 120 and 133 and delivery at term. 9 The only study that examined brain weights in sheep involved direct fetal administration of corticosteroids. Continuous infusion of catheterized animals for 60 hours from day 128 and delivery at 130 days showed a significant reduction in brain weight. 25 None of those large animal studies used an injection protocol that conformed to clinical practice.
The present study's protocol closely reflects treatment in human pregnancy (Table 4). Several of our measurements showed that single and repeated doses of corticosteroids slowed brain growth in sheep by term. The National Institutes of Health consensus on single corticosteroid treatment showed no long-term deleterious neurological or cognitive effects in humans. 5 If sheep are accurate models for humans, we suggest that effects of single courses of corticosteroids on sheep will be recovered in the long term. Studies addressing that issue are currently under way.
We have yet to determine whether effects of repeated corticosteroids on brain growth in our study are relevant to humans. A recent study in infants born at less than 33 weeks and observed to 3 years of age found that repeated corticosteroid doses significantly reduced birth weights and head circumferences by as much as 9% and 4%, respectively. 26 Behavioral problems increased with increasing corticosteroid treatment, assessed by externalizing behavior on the Child Behavior Checklist and the Distractibility scale on the Parenting Stress Index (French NP, Hagan R, Evans SF, Godfrey M, Newnham JP. Repeated antenatal corticosteroids (CS): Behaviour outcomes in a regional population of very preterm (VP, < 33w) infants [abstract]. Pediatr Res 1998;43:214). Further studies need to establish the relative risks and benefits of clinical antenatal corticosteroid use.
At the mechanistic level, glucocorticoids are powerful regulators of differentiation and maturation; therefore, administration of exogenous corticosteroids can alter brain development. 27 For example, in rats hydrocortisone depresses the activity of thymidine kinase activity, 28 an enzyme that regulates the rate of DNA synthesis and the production of nucleotides. A reduction in thymidine kinase activity would result in decreased cell division and delays in brain growth. Glucocorticoids regulate maturation of oligodendrocytes, 29 which produce myelin in the central nervous system, and they regulate production of key components of myelin, such as cerebrosides, proteolipid protein, and myelin basic protein. 30 It was shown that corticosteroids arrest myelination of the optic nerve. 8,13 Reductions in cell division and myelination of fiber tracts would contribute to decreased brain weights reported here and in other studies. 7,9
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