Objective
We investigated effects of 3 weekly courses of fetal betamethasone (βM) on motivation and cognition in juvenile baboon offspring utilizing the Cambridge Neuropsychological Test Automated Battery.
Study Design
Pregnant baboons ( Papio species) received 2 injections of saline control or 175 μg/kg βM 24 hours apart at 0.6, 0.65, and 0.7 gestation. Offspring (saline control female, n = 7 and saline control male, n = 6; βM female [FβM], n = 7 and βM male [MβM], n = 5) were studied at 2.6-3.2 years with a progressive ratio test for motivation, simple discriminations and reversals for associative learning and rule change plasticity, and an intra/extradimensional set-shifting test for attention allocation.
Results
βM exposure decreased motivation in both sexes. In intra/extradimensional testing, FβM made more errors in the simple discrimination reversal (mean difference of errors [FβM – MβM] = 20.2 ± 9.9; P ≤ .05), compound discrimination (mean difference of errors = 36.3 ± 17.4; P ≤ .05), and compound reversal (mean difference of errors = 58 ± 23.6; P < .05) stages as compared to the MβM offspring.
Conclusion
This central nervous system developmental programming adds growing concerns of long-term effects of repeated fetal synthetic glucocorticoid exposure. In summary, behavioral effects observed show sex-specific differences in resilience to multiple fetal βM exposures.
Fetal life is a period of intensive developmental plasticity during which an orchestrated and interactive pattern of organ development occurs. Postnatal survival depends on an adequate level of maturation of key systems such as the lung. In sheep, the prenatal rise in fetal cortisol that occurs in the final 20 days of gestation has been shown to be essential for terminal differentiation of the fetal lung. Administration of synthetic glucocorticoids (sGC) to pregnant women threatening premature labor has clearly proven to be beneficial in enhancing survival of offspring by accelerating lung maturation and decreasing the incidence of neonatal morbidity and mortality. However, several studies by others and ourselves in rodents, sheep, and baboons have shown that fetal exposure to sGC in the dosing regimen administered clinically have unwanted effects on the development of key fetal systems such as the peripheral vasculature, endocrine glands, and brain. Alterations in the trajectory of development are inevitable since the very purpose of the treatment is to produce circulating fetal glucocorticoid (GC) levels that are higher than those appropriate for the current stage of maturation of key fetal organs. There are limited findings on the long-term clinical effects of betamethasone (βM), a common sGC, on neurological development and cognition. Although no rigorous studies have been conducted on GC effects in human beings, a recent paper negatively correlates maternal stress and amniotic cortisol with behavior/cognitive outcomes in infants. To date there have been no studies to evaluate postnatal effects of sGC administration to pregnant nonhuman primates when given by the intramuscular clinical route and in the doses used clinically.
In this study, we sought to investigate the effects of βM administration on cognitive function and motivation in juvenile baboons exposed prenatally to levels of GC that were inappropriate for the stage of gestation utilizing the Cambridge Neuropsychological Test Automated Battery (CANTAB) system. The behaviors assessed were motivation (progressive ratio task), associative learning (simple discrimination [SD] task), rule change flexibility (SD reversal [SR] task), selective attention (intradimensional [ID] set task), and attentional allocation (extradimensional [ED] set-shift task). These behaviors rely on higher cortical function and normally developed limbic components. Our experimental design based βM administration on National Institute of Child Health and Human Development recommendation for prevention of complications associated with newborn prematurity. The timing of maternal treatment corresponded to the human clinical protocol at weeks 26, 27, and 28 (term 40 weeks). As in the case of human pregnancy GC do not change the length of nonhuman primate pregnancy in contrast to other experimental species such as sheep and thus represent a translatable model to human fetuses exposed to sGC that deliver at term. Although records are difficult to obtain, some authorities indicate that in excess of 50% of human babies who are exposed to inappropriate amounts of GC as a result of this efficacious therapy deliver at term thus enhancing the value of this study.
Materials and Methods
Subjects
All animal procedures were performed in accordance with accepted standards of humane animal care, approved by the Southwest Foundation for Biomedical Research and University of Texas Health Science Center at San Antonio (UTHSCSA) Institutional Animal Care and Use Committee, and conducted in AAALAC Inc–approved facilities, UTHSCSA. Pregnant baboons ( Papio species, n = 25) 10-15 years of age from the colony maintained at the Southwest National Primate Research Center (San Antonio, TX) were housed in outdoor metal and concrete gang cages, each containing 10-16 females and 1 breeding male. Animals were observed 3 times per week for evaluation of perineal skin swelling. Gestational age was calculated, using estimated day of conception (the time of last maximal perineal skin swelling minus 2 days).
Normal baboon gestation lasts 184 days. At 90 days of gestation (0.5 gestation [0.5G]), pregnant baboons were weighed, underwent a detailed ultrasound examination, and were placed in individual indoor cages. Baboons were randomized to receive saline (n = 13) or βM phosphate (n = 12) (Celestan Solubile; Essex Pharma, Munich, Germany) in doses of 175 μg/kg –1 /d –1 , a weight-adjusted dose equivalent to 12 mg administered to a 70-kg woman. Treatments were administered intramuscularly once daily at 8 am at 111, 112, 118, 119, 125, and 126 days of gestation (approximately equivalent to 24 [0.6G], 26 [0.65G], and 28 [0.7G] weeks of human pregnancy). Mothers delivered spontaneously at full term and offspring were reared with their mother in group housing until young adolescence. To ensure that testing was conducted at the same stage of postnatal development, offspring (saline control female [FC], n = 7 and saline control male [MC], n = 6; or 175 μg/kg βM female [FβM], n = 7 and βM male [MβM], n = 5) were transferred to the UTHSCSA in 4 different cohorts of 5-7 subjects across 2 years and housed individually in sight of at least 1 other subject in the UTHSCSA Laboratory Animal Resources facility.
Subjects were behaviorally tested at 2.9 ± 0.03 years of age when males weighed between 7-12 kg and females weighed between 6-9 kg, all being within the normal weight for this age. Training and testing were conducted between 9 am and 4 pm, Monday through Friday. If an animal was tested in the morning (9-11:30 am), feeding occurred at 12 and 5 pm or if tested in the afternoon (1-4 pm), feeding occurred at 9 am and 5 pm. Nonhuman primate chow (2050 Teklad Global 20% protein, 11.3 kJ/g metabolizable energy; Harlan Laboratories, Houston, TX) was given no less than 4 hours preceding behavioral sessions and at the end of the day (5 pm) with the exception of the progressive ratio task. For the progressive ratio task, each subject was fed 2 hours before the task was administered regardless of when the animal was tested and a second time at 5 pm. Daily chow rations were calculated prior to training by administering food ad libitum over a course of 2 weeks and measuring consumption. Each subject would then be fed half this amount twice per day over the course of the study (3 months). In this manner feed was adjusted to each individual subject and no refusal to eat was observed. Water was always available and fruit and vitamins supplemented the diet on Monday, Wednesday, and Friday. The light cycle was set with lights on at 7 am and off at 9 pm.
Equipment/CANTAB training and testing
Our use of the CANTAB system for baboon behavioral testing has been described in detail ( Appendix ).
Data analysis
Data are summarized with the mean ± SE. Mean contrasts with regard to treatment (control vs βM) and sex (female vs male) were performed using either a linear model or a repeated measures linear model with an autoregressive correlation structure to model the association between successive trials. The interaction of treatment and sex was included in all models. All subjects provided data for all tasks except 1 subject (a MC), which was excluded from participation in the intra-/extra-dimensional (IDED) attention set-shifting test for failing to reach this stage contemporaneously with the others. Software (SAS, version 9.2; SAS Institute, Cary, NC) was used to conduct analyses; statistical significance was set at P ≤ .05.
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