http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–5 ! 2015 Informa UK Ltd. DOI: 10.3109/14767058.2015.1035639
ORIGINAL ARTICLE
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Neonatal neuronal apoptosis after betamethasone administration in pregnant Wistar rats Marcelo Santucci Franc¸a1, Antonio Fernandes Moron1, Edward Araujo Ju´nior1, Marcelo Avedissian2, David Baptista Silva Pares1, Luciano Marcondes Marchado Nardozza1, Carolina Barros Jaqueta2, and Luiz Eugeˆnio Araujo Moraes Mello2 1
Department of Obstetrics, Fetal Medicine Discipline and 2Department of Physiology, Division of Neurophysiology, Paulista School of Medicine, Federal University of Sa˜o Paulo (EPM-UNIFESP), Sa˜o Paulo, SP, Brazil Abstract
Keywords
Objective: To analyze the apoptosis of cortical and hippocampal neurons in newborn following the intramuscular administration of betamethasone in pregnant Wistar rats. Methods: Betamethasone or placebo was administered to 10 pregnant rats. Subsequently, 98 newborns were analyzed in three different groups: therapeutic dose (TD, 20 mg/kg), triple therapeutic dose (3TD, 60 mg/kg), and nine times TD (9TD, 180 mg/kg). Forty-four newborns were injected with placebo (control subjects – CTR). Neuronal apoptosis was measured by immunofluorescence using the TUNEL assay. The one-way analysis of variance, Tukey–Kramer (parametric) test and Kruskal–Wallis (non-parametric) test were used for statistical analysis. Results: The CA1 area of the hippocampus of TD and 3TD groups showed significant differences from that of the CTR group (p50.001). Compared to the CTR group, there was increased neuronal apoptosis in the dentate gyrus (DG) of animals in TD and 3TD groups (p50.0001). There were no significant differences in CA2 and CA3 regions as well as in amygdala and cortex. Conclusion: Prenatal administration of betamethasone leads to significant changes in neuronal apoptosis in CA1 and DG regions.
Betamethasone, immunofluorescence, neonatal neuronal apoptosis, pregnancy, TUNEL assay, Wistar rats
Introduction Antenatal glucocorticoid (AG) treatment with betamethasone (BETA) is routinely used in women at risk of preterm delivery under 34 weeks of gestation [1]. BETA promotes fetal lung development between the 28th and 32nd weeks of gestation [2] and reduces mortality of the preterm infant after delivery [3]. Respiratory distress syndrome (RDS), a consequence of immature lung development, is the principal cause of early neonatal mortality and contributes significantly to the high costs of neonatal intensive care. Since preterm babies are at higher risks of neurological disabilities [4], strategies that reduce the risk of neonatal RDS in preterm delivery have received considerable attention [5]. A single course of AG reduces the risk of RDS from 40% to 21% in babies born before 32 weeks of gestation [2]. However, although AG administration is an effective strategy for reducing the adverse consequences of preterm birth and despite the postnatal intensive care and use of exogenous surfactants, neonatal morbidity has remained significantly Address for correspondence: Prof. Edward Araujo Ju´nior, PhD, Department of Obstetrics, Fetal Medicine Discipline, Paulista School of Medicine, Federal University of Sa˜o Paulo (EPM-UNIFESP), Rua Carlos Weber, 956, apto. 113 Visage, Sa˜o Paulo, SP CEP 05303, Brazil. Tel/Fax: +55-11-37965944. E-mail: [email protected]
History Received 16 December 2014 Accepted 26 March 2015 Published online 29 April 2015
high [5]. AG reduces the risks of neonatal mortality, intraventricular hemorrhage and minimizes the need for surfactant therapy [2]. Corticosteroids are known to inhibit cellular growth and DNA replication. Studies have shown that maternal administration of pharmacological doses of steroids inhibits fetal growth [6] and alters gene expression in the prefrontal cortex [7]. In lambs, four doses of BETA administered to the ewe reduce the birth weight [8]. Randomized trials have found no apparent neurologic or cognitive effects in children treated with a single dose of AG [9]. Other studies have found that repeated doses of steroids have harmful effects on neuronal myelination [10]. The current trend in clinical practice is to administer repeated doses of AG, which is associated with head circumference reduction [12]. It has been reported that acutely elevated glucocorticoid levels impairs declarative memory retrieval in children [11]. In animals, multiple courses of AG causes delayed growth [8] and a reduction in the number of neurons and corticosteroid receptors in the hippocampus [13], suggesting that AG may have deleterious effects in developing nervous system. During gestation, endogenous stress could lead to an increase in the serum levels of glucocorticoids and affect central nervous system development. It has been reported that
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in humans, maternal stress during the third trimester is associated with intra-uterine growth restriction (IUGR) [17] and behavioral abnormalities [14]. In animals, antenatal BETA administration delays myelination of the corpus callosum [15], reduces anxiety [16], attenuates hippocampal glucocorticoid receptor gene expression [13] and causes IUGR [8]. In IUGR fetus, BETA induces brain injury by reducing carotid blood flow [11]. Despite these studies, the effects of BETA on the developing nervous system remain poorly understood. The objective of this study was to evaluate neuronal apoptosis in newborns following intramuscular administration of BETA in pregnant Wistar rats.
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Methods Animals Female Wistar EPM-1 (n ¼ 24) rats (250–280 g) were obtained from the bioterium of the Federal University of Sa˜o Paulo (UNIFESP) and were bred with male (n ¼ 24) Wistar EPM-1 rats. Animals were housed individually in separate cages at 21 C under a 12/12 light-dark cycle. Water and food were provided ad libitum. Rats were housed in the same sex pairs in 45 45 25 cm plastic cages until delivery. The study was approved by the Research Ethics Committee of the UNIFESP (157/03). Breeding, antenatal treatment Female rats were bred with males for 12 h (at night). After this period, the presence of sperm in the vagina indicated day 1 of pregnancy when the males were immediately separated from the females. The inseminated female rats were individually housed in cages with clean bedding. Pregnant rats received antenatal BETA treatment on the 13th and 14th (second third) day of pregnancy. The antenatal treatment consisted of a single administration of BETA at a dose of 0.2 mg/kg (TD) (n ¼ 5), 0.6 mg/kg (3TD) (n ¼ 4), 1.8 mg/kg (9TD) (n ¼ 6) or a vehicle solution of 45% 2-hydroxypropil-b-cyclodextran (n ¼ 4). All treatments were administered intramuscularly to the right-hind limb at 11:00 a.m. every day. The total volume injected was between 0.1 mL and 0.3 mL. The dosage of corticosteroids administered was similar to that used clinically in humans [18] over a 24-h period. The offspring resided in the cages with the dam until birth. The total number of pups in each litter, live births, stillbirths and early neonatal deaths (born alive but had died by postnatal at day 1) were assessed. The half lethal dose (LD50) of this drug for the animals was 6.5 mg/kg/dose. Even at nine times the therapeutic dose, the daily dose of BETA administered was only 1.8 mg/kg. Thus, the rats were not exposed to LD50 dose of BETA. The pregnant female rats delivered naturally (without the researcher’s interference). After 48 h of life (PND2), the newborns were decapitated and the encephalon was retrieved. The TUNEL immunofluorescence assay was used to assess the apoptosis. Neurons were counted manually.
J Matern Fetal Neonatal Med, Early Online: 1–5
paraformaldehyde/0.1 M phosphate buffer saline (PBS, pH 7.4) for 48 h until dehydration and cryosection. The encephalons were sectioned into 40-mm coronal blocks, starting from the knee of the corpus callosum, at 19 C. The sections were stored in anti-freeze solution until immunofluorescence staining. Apoptosis assay The deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay kit (Roche Molecular Biochemical’sÕ , Mannheim, Germany) was used to assess neuronal apoptosis according to the manufacturer’s instructions [19–22]. After immunohistochemistry, stained area was calculated by semi-quantitative methods. Typically, the brain tissue was fixed using paraformaldehyde (4%, pH 7.4) for 2 h, rinsed with phosphate buffer saline (PBS; 0.1 M, pH 7.4) containing sucrose (15%) and was stored frozen in liquid nitrogen before sectioning. Continuous serial frontal sections (40 mm thickness) were prepared, conditioned in anti-freeze solution, washed thrice with PBS for 10 min and permeabilized in 2N HCL for 60 min at 60 C in ‘‘free-float’’ mode. The sections were then washed with PBS and mounted on sheets. The mounted sections were dried for 2 h. Following this, the sections were incubated with terminal deoxinucleotidil transferase (TdT) and a mixture of nucleotide fluorescein isothiocyanate (FITC)-conjugated dUTP in a humidified atmosphere for 60 min at 37 C. The labeled sections were washed thrice with PBS for 2 min each and the sheets were sealed with DPX. Following this, the labeled samples were analyzed under a fluorescence microscope (450 to 550 nm). Apoptotic (characterized by the presence of a dark nuclear precipitate) and non-apoptotic (no staining or diffuse cytoplasmic staining) cells in a chosen microscopic field (containing at least 100 cells, predetermined as 22 500 mm2) were counted manually. The following anatomical structures were analyzed: hippocampus [Dentate Gyrus (DG sup and DG), CA1, CA1 medial portion, CA2 and CA3], amygdala and sagital cortex (CTX). The cortex was subdivided into CTX 12:00, CTX 1:00, CTX 2:00, CTX 3:00, CTX 4:00 and CTX 5:00 as reported earlier. The results were expressed as average number of apoptotic cells in a field with standard deviation. Manual counting of cells permitted reanalysis of the data whenever necessary (Figure 1). Only four to six pups per litter were used to minimize the ‘‘within litter’’ effects on experimental results [23]. Statistical analysis Parametric data were analyzed by one-way ANOVA and the Tukey–Kramer test and non-parametric data were analyzed using the Kruskal–Wallis test. The PASW (version 18.0, SPSS Inc., Chicago, IL) and GraphPad Prism softwares (version 6.0, GraphPad Software Inc., San Diego, CA) were used for statistical analysis.
Results Tissue preparation and histology
Fetal mortality after conception
On PND2, all pups were decapitated and their encephalons were retrieved and stored at 4C in 1%
The results (Table 1) showed that the group 9TD had the highest rate of perinatal mortality (97%). The mortality rates
Neonatal neuronal apoptosis
DOI: 10.3109/14767058.2015.1035639
in the control subjects (CTR) and 3TD groups were 2.3% and 7.3%, respectively. Thus, the mortality rate in 9TD was significantly higher than that in other groups (p50.001, by Fisher’s exact test). Although the mortality rate in the TD group was less than that in the CTR and 3DT groups, this difference was statistically insignificant (p ¼ 0.073).
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Neuronal cell count Due to high mortality rate, the neuronal cell count of the 9TD group was not compared with that of other groups. The extent of apoptosis in the hippocampus was assessed by calculating the average (standard deviation) number of apoptotic neurons per field, whereas for midbrain, the median (maximum–minimum) number of apoptotic neurons per field was estimated (Table 2). Multiple comparisons using the Tukey–Kramer test revealed significant difference between the CA1 regions of the TD and 3TD groups and that of the CTR group (Figure 2). The average the number of neurons was significantly different (p ¼ 0.011 by one-way ANOVA) (Figure 3). Compared with the TD and 3TD groups, the CTR group showed only a small amount of apoptotic neurons. The number of apoptotic neurons increased with the dose of BETA. Besides, the group DT did not differ of the CTR or the 3DT group. Other hippocampal areas (CA1 medial, CA2, CA3 and DG sup.) did not differ significantly (p40.05). The Kruskal–Wallis test demonstrated no statistically significant differences between the number of apoptotic neurons in the cortex and amygdala of the groups.
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reduced the number of apoptotic neurons in the CA1 region and increased neuronal cell death in the DG. Previous studies have found reduced expression of glucocorticoids receptors (GR) following adrenalectomy [24], so inferences are allowed due to action of the BETA at encephalon. The medication effects are antagonistic and can be evidenced in CA1 and DG inf. In DG inf., the neuronal mortality increased in the treated groups and in CA1 decreased the apoptosis index. This compensatory mechanism observed in the CA1 region after the administration of BETA can be explained by the low numbers of GR found in this area. The increased neuronal cell death observed in DG following BETA administration is consistent with the earlier report [24] of reduced apoptosis in adult animals during periods of low level of steroids and after adrenalectomy. The increased apoptosis in DG and reduced cell death in the CA1 region point toward the existence of an indirect compensatory pathway (statistically significant difference). A new balance was reached in hippocampus distinct areas, keeping alive a larger neuronal contingent. There are no evidences in the literature that the decreasing of the apoptosis in CA1 is due to direct action of BETA, but the results suggests that this mortality is a consequence of a previous lesion in DG. In humans, AG use do not cause neurological disabilities after birth [2,3]; however, it is larger the number of
Table 1. Perinatal result for each group.
Discussion
Groups
We analyzed various regions of the brains of BETA- and placebo-treated animals for differences in the number of apoptotic neurons. Our results showed that the groups differed in the number of apoptotic neurons in the CA1 and DG regions of their hippocampus. The administration of BETA
CTR TD 3TD 9TD
Injected animals
Number of fetuses
Number of newborns
Number of newborns/ pregnancy
4 5 4 6
42 48 41 60
41 48 38 2
10.25 9.6 9.5 0.033*
*Fisher’s exact test.
Figure 1. Selected field of control group (CTR) (left), which was treated with the therapeutic dose (TD) of BETA (right). The magenta images indicate apoptotic neurons. The triangles in the vertices represent the boundary of the area and the number of apoptotic cells in the selected field. The yellow scale indicates the distance of 50 mm, which was used as the standard for the calculation of the area.
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J Matern Fetal Neonatal Med, Early Online: 1–5
Table 2. Neuronal apoptosis in hippocampus and midbrain. Groups Hippocampus CA1 DG inf. CA1 medial CA2 CA3
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DG sup. Midbrain CTX 12 h CTX 1 h CTX 2 h CTX 3 h CTX 5 h Amygdala
CTR
TD
3TD
p
61.6 (13.8) N¼6 17.6 (4.3) N¼4 45.0 (4.6) N¼3 47.2 (6.5) N¼6 34.6 (10.1) N¼5 45.6 (17.6) N¼4 50 (23–130) N¼5 60 (48–107) N¼5 65 (50–72) N¼4 71 (43–90) N¼4 43 (43–43) N¼1 48 (34–50) N¼3
17.7 (7.1) N¼5 26.3 (4.0) N¼3 42.2 (11.2) N¼4 43.6 (14.3) N¼5 35.0 (19.3) N¼5 25.4 (10.2) N¼5 57 (54–58) N¼3 60 (58–66) N¼3 63 (53–90) N¼4 57 (57–57) N¼1 528 (528–528) N¼1 57 (36–61) N¼3
40.9 (10.2) N¼5 32.2 (8.0) N¼5 39.0 (4.2) N¼4 47.0 (15.8) N¼6 41.7 (9.9) N¼6 37.0 (20.8) N¼4 35 (42–73) N¼6 64 (57–71) N¼2 75 (43–102) N¼5 60 (40–78) N¼3 58 (53–62) N¼2 52 (46–85) N¼6
0.001y 0.011* 0.47* 0.84* 0.63* 0.31* 0.96y 0.91y 0.62y 0.49y 0.25y 0.37y
*Tukey–Kramer. yKruskall–Wallis.
Figure 2. Average ± standard deviation from CA1 by group. (*) indicates significant difference between experimental (TD and 3TD) group and control (CTR) group.
Figure 3. Average ± standard deviation from DG by group. (*) indicates significant? difference between the 3TD group and the control (CTR) group.
psychomotor disturbances involving AG and prematurity [25] or with the pregnancy and stress, as depression [28], attention deficit hyperactivity disorder (ADHD) [25,27,28], schizophrenia [29,30] and epilepsy [31]. There is a positive correlation between the reduction of the encephalon volume of masculine children, ADHD and AG use [27]. The smaller volume of caudate/putamen in human adult with ADHD, described by Castellanos and Acosta [27] can be associated to the decreasing encephalic volume, after AG [1]. It is suggested the volumetric encephalic reduction described by Liggins and Howie [1] and Castellanos and Acosta [27] can be associated with this hippocampus pattern apoptosis and co-made responsible for the genesis of this behavioral alteration [26]. Caudate/ putamen is predominantly responsible for the mental processes related to the prefrontal cortex and very wide connections with hippocampus. It is likely that neuronal cell death contributes to ADHD. The ADHD genesis is correlated with smoking, trauma, stress, alcoholism [28] and AG in premature children [14] or children with stress pregnancy [25]. The opposing effects of BETA on neuronal cell death in DG and CA1 regions can be seen as a result of compensatory mechanisms triggered to minimize the potentially harmful effects of the loss of DG neurons [26], which is likely responsible for the behavioral alterations observed in users of AG. These behavioral changes may reflect lesion in areas such as DG, and of the incapacity of biological reaction of areas as CA1. Were used genetically identical animals, with standard results; however, according to the author’s opinion, in human individuals, with larger genetic and environmental variability, there were different answers after different stimulation. The apoptosis mechanisms could facilitate epileptogenesis in the temporal lobe. In another word, areas with apoptosis increasing, as the DG inf. after AG, can be more susceptible to the propagation of the electric pulse, due to insufficient inhibitions. Together with the findings of earlier studies [24], our results demonstrate that DG responds differently to the absence or excess of glucocorticoids, increased apoptosis in DG and the reduced apoptosis in CA1, demonstrated in this study; and the association between AG and the epileptogenesis of temporal lobe for the decreasing of the epileptic threshold in experimentation animals [31]. The high number of GR in the DG [24] could lead to increased, reducing the neuronal inhibition and facilitating epileptogenesis after appropriate stimulation [31]. In summary, our results showed significant difference in neuronal apoptosis in CA1 and DG regions of the offspring following the administration of BETA in pregnant rats. Such
DOI: 10.3109/14767058.2015.1035639
differences in the neuronal apoptosis between DG and CA1 regions may contribute to the development of ADHD, schizophrenia and epilepsy.
Acknowledgements This work was supported by FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo) Grant No. 04/00877-0.
Declaration of interest The authors declare no conflicts of interests.
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References 1. Liggins GC, Howie RN. A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants. Pediatrics 1972;50:515–25. 2. Crowley P. Prophylactic corticosteroids for preterm birth. Cochrane Database Syst Rev 2000;(2):CD000065. 3. Crowther CA, Harding J. Repeat doses of prenatal corticosteroids for women at risk of preterm birth for preventing neonatal respiratory disease. Cochrane Database Syst Rev 2003;(3):CD003935. 4. Paro-Panjan D, Kodri J, Sustersic B. Association between neurological signs and developmental outcome: pilot results in preterm group. Croat Med J 2009;50:345–50. 5. Soll R, Morley CJ. Prophylactic versus selective use of surfactant in preventing morbidity and mortality in preterm infants. Cochrane Database Syst Rev 2000;(2):CD000510. 6. Wapner RJ, Sorokin Y, Thom EA, et al. Single versus weekly courses of antenatal corticosteroids: evaluation of safety and efficacy. Am J Obstet Gynecol 2006;195:633–42. 7. Diaz Heijtz R, Fuchs E, Feldon J, et al. Effects of antenatal dexamethasone treatment on glucocorticoid receptor and calcyon gene expression in the prefrontal cortex of neonatal and adult common marmoset monkeys. Behav Brain Funct 2010;6:18. 8. Ikegami M, Jobe AH, Newnham J, et al. Repetitive prenatal glucocorticoids improve lung function and decrease growth in preterm lambs. Am J Respir Crit Care Med 1997;156:178–84. 9. Murphy DJ, Caukwell S, Joels LA, Wardle P. Cohort study of the neonatal outcome of twin pregnancies that were treated with prophylactic or rescue antenatal corticosteroids. Am J Obstet Gynecol 2002;187:483–8. 10. Dunlop SA. Repeated prenatal corticosteroids delay myelination in the ovine central nervous system. J Mater Fetal Med 1997;6: 309–13. 11. De Quervain DJ, Henke K, Aerni A, et al. Glucocorticoid-induced impairment of declarative memory retrieval is associated with reduced blood flow in the medial temporal lobe. Eur J Neurosci 2003;17:1296–302. 12. French NP, Hagan R, Evans SF, et al. Repeated antenatal corticosteroids: size at birth and subsequent development. Am J Obstet Gynecol 1999;180:114–21. 13. Levitt NS, Lindsay RS, Holmes MC, Seckl JR. Dexamethasone in the last week of pregnancy attenuates hippocampal glucocorticoid receptor gene expression and elevates blood pressure in the adult offspring in the rat. Neuroendocrinology 1996;64:412–18. 14. French NP, Hagan R, Evans SF, et al. Repeated antenatal corticosteroids: effects on cerebral palsy and childhood behavior. Am J Obstet Gynecol 2004;190:588–95.
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15. Huang WL, Harper CG, Evans SF, et al. Repeated prenatal corticosteroid administration delays myelination of the corpus callosum in fetal sheep. Int J Dev Neurosci 2001;19:415–25. 16. Rayburn WF, Christensen HD, Gonzalez CL. A placebo-controlled comparison between betamethasone and dexamethasone for fetal maturation: differences in neurobehavioral development of mice offspring. Am J Obstet Gynecol 1997;176:842–50. 17. Miller SL, Chai M, Loose J, et al. The effects of maternal betamethasone administration on the intrauterine growth-restricted fetus. Endocrinology 2007;148:1288–95. 18. Crane J, Armson A, Brunner M, et al. Antenatal corticosteroid therapy for fetal maturation. J Obstet Gynaecol Can 2003;25: 45–52. 19. Smith C, Yu YX, Kulka M, Aurelian L. A novel human gene similar to the PK coding domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) codes for a serine-threonine PK and is expressed in melanoma cells. J Biol Chem 2000;275:25690–9. 20. Perkins D, Pereira EE, Aurelian L. The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) functions as a dominant regulator of apoptosis in hippocampal neurons involving activation of the ERK survival pathway and upregulation of the antiapoptotic protein Bag1. J Virol 2003;77:1292–305. 21. Perkins D, Pereira EF, Gober M, et al. The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) blocks apoptosis in hippocampal neurons involving activation of the MEK/mitogen activated protein kinase survival pathway. J Virol 2002;76: 1435–49. 22. Gober MD, Smith CC, Ueda K, et al. Forced expression of the H11 heat shock protein can be regulated by DNA methylation and trigger apoptosis in human cells. J Biol Chem 2003;26;278:37600–9. 23. Zorrilla EP. Multiparous species present problems (and possibilities) to developmentalists. Dev Psychobiol 1997;30:141–50. 24. Covolan L, Ribeiro LT, Longo BM, Mello LE. Cell damage and neurogenesis in the dentate granule cell layer of adult rats after pilocarpine- or kainate-induced status epilepticus. Hippocampus 2000;10:169–80. 25. McGrath MM, Sullivan M, Devin J, et al. Early precursors of low attention and hyperactivity in a preterm sample at age four. Issues Compr Pediatr Nurs 2005;28:1–15. 26. Hu Z, Yuri K, Ichikawa T, Kawata M. Exposure of postnatal rats to glucocorticoids suppresses the development of choline acetyltransferase-immunoreactive neurons: role of adrenal steroids in the development of forebrain cholinergic neurons. J Chem Neuroanat 1996;10:1–10. 27. Castellanos FX, Acosta MT. The neuroanatomy of attention-deficit/ hyperactivity disorder. Rev Neurol 2004;38(Suppl 1):S131–6. 28. Linnet KM, Dalsgaard S, Obel C, et al. Maternal lifestyle factors in pregnancy risk of attention deficit hyperactivity disorder and associated behaviors: review of the current evidence. Am J Psychiatr 2003;160:1028–40. 29. Koenig JI, Elmer GI, Shepard PD, et al. Prenatal exposure to a repeated variable stress paradigm elicits behavioral and neuroendocrinological changes in the adult offspring: potential relevance to schizophrenia. Behav Brain Res 2005;156:251–61. 30. Hauser J, Feldon J, Pryce CR. Prenatal dexamethasone exposure, postnatal development, and adulthood prepulse inhibition and latent inhibition in Wistar rats. Behav Brain Res 2006;175:51–61. 31. Young NA, Teskey GC, Henry LC, Edwards HE. Exogenous antenatal glucocorticoid treatment reduces susceptibility for hippocampal kindled and maximal electroconvulsive seizures in infant rats. Exp Neurol 2006;198:303–12.
ORIGINAL ARTICLE
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Nyu Medical Center on 05/07/15 For personal use only.
Neonatal neuronal apoptosis after betamethasone administration in pregnant Wistar rats Marcelo Santucci Franc¸a1, Antonio Fernandes Moron1, Edward Araujo Ju´nior1, Marcelo Avedissian2, David Baptista Silva Pares1, Luciano Marcondes Marchado Nardozza1, Carolina Barros Jaqueta2, and Luiz Eugeˆnio Araujo Moraes Mello2 1
Department of Obstetrics, Fetal Medicine Discipline and 2Department of Physiology, Division of Neurophysiology, Paulista School of Medicine, Federal University of Sa˜o Paulo (EPM-UNIFESP), Sa˜o Paulo, SP, Brazil Abstract
Keywords
Objective: To analyze the apoptosis of cortical and hippocampal neurons in newborn following the intramuscular administration of betamethasone in pregnant Wistar rats. Methods: Betamethasone or placebo was administered to 10 pregnant rats. Subsequently, 98 newborns were analyzed in three different groups: therapeutic dose (TD, 20 mg/kg), triple therapeutic dose (3TD, 60 mg/kg), and nine times TD (9TD, 180 mg/kg). Forty-four newborns were injected with placebo (control subjects – CTR). Neuronal apoptosis was measured by immunofluorescence using the TUNEL assay. The one-way analysis of variance, Tukey–Kramer (parametric) test and Kruskal–Wallis (non-parametric) test were used for statistical analysis. Results: The CA1 area of the hippocampus of TD and 3TD groups showed significant differences from that of the CTR group (p50.001). Compared to the CTR group, there was increased neuronal apoptosis in the dentate gyrus (DG) of animals in TD and 3TD groups (p50.0001). There were no significant differences in CA2 and CA3 regions as well as in amygdala and cortex. Conclusion: Prenatal administration of betamethasone leads to significant changes in neuronal apoptosis in CA1 and DG regions.
Betamethasone, immunofluorescence, neonatal neuronal apoptosis, pregnancy, TUNEL assay, Wistar rats
Introduction Antenatal glucocorticoid (AG) treatment with betamethasone (BETA) is routinely used in women at risk of preterm delivery under 34 weeks of gestation [1]. BETA promotes fetal lung development between the 28th and 32nd weeks of gestation [2] and reduces mortality of the preterm infant after delivery [3]. Respiratory distress syndrome (RDS), a consequence of immature lung development, is the principal cause of early neonatal mortality and contributes significantly to the high costs of neonatal intensive care. Since preterm babies are at higher risks of neurological disabilities [4], strategies that reduce the risk of neonatal RDS in preterm delivery have received considerable attention [5]. A single course of AG reduces the risk of RDS from 40% to 21% in babies born before 32 weeks of gestation [2]. However, although AG administration is an effective strategy for reducing the adverse consequences of preterm birth and despite the postnatal intensive care and use of exogenous surfactants, neonatal morbidity has remained significantly Address for correspondence: Prof. Edward Araujo Ju´nior, PhD, Department of Obstetrics, Fetal Medicine Discipline, Paulista School of Medicine, Federal University of Sa˜o Paulo (EPM-UNIFESP), Rua Carlos Weber, 956, apto. 113 Visage, Sa˜o Paulo, SP CEP 05303, Brazil. Tel/Fax: +55-11-37965944. E-mail: [email protected]
History Received 16 December 2014 Accepted 26 March 2015 Published online 29 April 2015
high [5]. AG reduces the risks of neonatal mortality, intraventricular hemorrhage and minimizes the need for surfactant therapy [2]. Corticosteroids are known to inhibit cellular growth and DNA replication. Studies have shown that maternal administration of pharmacological doses of steroids inhibits fetal growth [6] and alters gene expression in the prefrontal cortex [7]. In lambs, four doses of BETA administered to the ewe reduce the birth weight [8]. Randomized trials have found no apparent neurologic or cognitive effects in children treated with a single dose of AG [9]. Other studies have found that repeated doses of steroids have harmful effects on neuronal myelination [10]. The current trend in clinical practice is to administer repeated doses of AG, which is associated with head circumference reduction [12]. It has been reported that acutely elevated glucocorticoid levels impairs declarative memory retrieval in children [11]. In animals, multiple courses of AG causes delayed growth [8] and a reduction in the number of neurons and corticosteroid receptors in the hippocampus [13], suggesting that AG may have deleterious effects in developing nervous system. During gestation, endogenous stress could lead to an increase in the serum levels of glucocorticoids and affect central nervous system development. It has been reported that
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in humans, maternal stress during the third trimester is associated with intra-uterine growth restriction (IUGR) [17] and behavioral abnormalities [14]. In animals, antenatal BETA administration delays myelination of the corpus callosum [15], reduces anxiety [16], attenuates hippocampal glucocorticoid receptor gene expression [13] and causes IUGR [8]. In IUGR fetus, BETA induces brain injury by reducing carotid blood flow [11]. Despite these studies, the effects of BETA on the developing nervous system remain poorly understood. The objective of this study was to evaluate neuronal apoptosis in newborns following intramuscular administration of BETA in pregnant Wistar rats.
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Methods Animals Female Wistar EPM-1 (n ¼ 24) rats (250–280 g) were obtained from the bioterium of the Federal University of Sa˜o Paulo (UNIFESP) and were bred with male (n ¼ 24) Wistar EPM-1 rats. Animals were housed individually in separate cages at 21 C under a 12/12 light-dark cycle. Water and food were provided ad libitum. Rats were housed in the same sex pairs in 45 45 25 cm plastic cages until delivery. The study was approved by the Research Ethics Committee of the UNIFESP (157/03). Breeding, antenatal treatment Female rats were bred with males for 12 h (at night). After this period, the presence of sperm in the vagina indicated day 1 of pregnancy when the males were immediately separated from the females. The inseminated female rats were individually housed in cages with clean bedding. Pregnant rats received antenatal BETA treatment on the 13th and 14th (second third) day of pregnancy. The antenatal treatment consisted of a single administration of BETA at a dose of 0.2 mg/kg (TD) (n ¼ 5), 0.6 mg/kg (3TD) (n ¼ 4), 1.8 mg/kg (9TD) (n ¼ 6) or a vehicle solution of 45% 2-hydroxypropil-b-cyclodextran (n ¼ 4). All treatments were administered intramuscularly to the right-hind limb at 11:00 a.m. every day. The total volume injected was between 0.1 mL and 0.3 mL. The dosage of corticosteroids administered was similar to that used clinically in humans [18] over a 24-h period. The offspring resided in the cages with the dam until birth. The total number of pups in each litter, live births, stillbirths and early neonatal deaths (born alive but had died by postnatal at day 1) were assessed. The half lethal dose (LD50) of this drug for the animals was 6.5 mg/kg/dose. Even at nine times the therapeutic dose, the daily dose of BETA administered was only 1.8 mg/kg. Thus, the rats were not exposed to LD50 dose of BETA. The pregnant female rats delivered naturally (without the researcher’s interference). After 48 h of life (PND2), the newborns were decapitated and the encephalon was retrieved. The TUNEL immunofluorescence assay was used to assess the apoptosis. Neurons were counted manually.
J Matern Fetal Neonatal Med, Early Online: 1–5
paraformaldehyde/0.1 M phosphate buffer saline (PBS, pH 7.4) for 48 h until dehydration and cryosection. The encephalons were sectioned into 40-mm coronal blocks, starting from the knee of the corpus callosum, at 19 C. The sections were stored in anti-freeze solution until immunofluorescence staining. Apoptosis assay The deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay kit (Roche Molecular Biochemical’sÕ , Mannheim, Germany) was used to assess neuronal apoptosis according to the manufacturer’s instructions [19–22]. After immunohistochemistry, stained area was calculated by semi-quantitative methods. Typically, the brain tissue was fixed using paraformaldehyde (4%, pH 7.4) for 2 h, rinsed with phosphate buffer saline (PBS; 0.1 M, pH 7.4) containing sucrose (15%) and was stored frozen in liquid nitrogen before sectioning. Continuous serial frontal sections (40 mm thickness) were prepared, conditioned in anti-freeze solution, washed thrice with PBS for 10 min and permeabilized in 2N HCL for 60 min at 60 C in ‘‘free-float’’ mode. The sections were then washed with PBS and mounted on sheets. The mounted sections were dried for 2 h. Following this, the sections were incubated with terminal deoxinucleotidil transferase (TdT) and a mixture of nucleotide fluorescein isothiocyanate (FITC)-conjugated dUTP in a humidified atmosphere for 60 min at 37 C. The labeled sections were washed thrice with PBS for 2 min each and the sheets were sealed with DPX. Following this, the labeled samples were analyzed under a fluorescence microscope (450 to 550 nm). Apoptotic (characterized by the presence of a dark nuclear precipitate) and non-apoptotic (no staining or diffuse cytoplasmic staining) cells in a chosen microscopic field (containing at least 100 cells, predetermined as 22 500 mm2) were counted manually. The following anatomical structures were analyzed: hippocampus [Dentate Gyrus (DG sup and DG), CA1, CA1 medial portion, CA2 and CA3], amygdala and sagital cortex (CTX). The cortex was subdivided into CTX 12:00, CTX 1:00, CTX 2:00, CTX 3:00, CTX 4:00 and CTX 5:00 as reported earlier. The results were expressed as average number of apoptotic cells in a field with standard deviation. Manual counting of cells permitted reanalysis of the data whenever necessary (Figure 1). Only four to six pups per litter were used to minimize the ‘‘within litter’’ effects on experimental results [23]. Statistical analysis Parametric data were analyzed by one-way ANOVA and the Tukey–Kramer test and non-parametric data were analyzed using the Kruskal–Wallis test. The PASW (version 18.0, SPSS Inc., Chicago, IL) and GraphPad Prism softwares (version 6.0, GraphPad Software Inc., San Diego, CA) were used for statistical analysis.
Results Tissue preparation and histology
Fetal mortality after conception
On PND2, all pups were decapitated and their encephalons were retrieved and stored at 4C in 1%
The results (Table 1) showed that the group 9TD had the highest rate of perinatal mortality (97%). The mortality rates
Neonatal neuronal apoptosis
DOI: 10.3109/14767058.2015.1035639
in the control subjects (CTR) and 3TD groups were 2.3% and 7.3%, respectively. Thus, the mortality rate in 9TD was significantly higher than that in other groups (p50.001, by Fisher’s exact test). Although the mortality rate in the TD group was less than that in the CTR and 3DT groups, this difference was statistically insignificant (p ¼ 0.073).
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Neuronal cell count Due to high mortality rate, the neuronal cell count of the 9TD group was not compared with that of other groups. The extent of apoptosis in the hippocampus was assessed by calculating the average (standard deviation) number of apoptotic neurons per field, whereas for midbrain, the median (maximum–minimum) number of apoptotic neurons per field was estimated (Table 2). Multiple comparisons using the Tukey–Kramer test revealed significant difference between the CA1 regions of the TD and 3TD groups and that of the CTR group (Figure 2). The average the number of neurons was significantly different (p ¼ 0.011 by one-way ANOVA) (Figure 3). Compared with the TD and 3TD groups, the CTR group showed only a small amount of apoptotic neurons. The number of apoptotic neurons increased with the dose of BETA. Besides, the group DT did not differ of the CTR or the 3DT group. Other hippocampal areas (CA1 medial, CA2, CA3 and DG sup.) did not differ significantly (p40.05). The Kruskal–Wallis test demonstrated no statistically significant differences between the number of apoptotic neurons in the cortex and amygdala of the groups.
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reduced the number of apoptotic neurons in the CA1 region and increased neuronal cell death in the DG. Previous studies have found reduced expression of glucocorticoids receptors (GR) following adrenalectomy [24], so inferences are allowed due to action of the BETA at encephalon. The medication effects are antagonistic and can be evidenced in CA1 and DG inf. In DG inf., the neuronal mortality increased in the treated groups and in CA1 decreased the apoptosis index. This compensatory mechanism observed in the CA1 region after the administration of BETA can be explained by the low numbers of GR found in this area. The increased neuronal cell death observed in DG following BETA administration is consistent with the earlier report [24] of reduced apoptosis in adult animals during periods of low level of steroids and after adrenalectomy. The increased apoptosis in DG and reduced cell death in the CA1 region point toward the existence of an indirect compensatory pathway (statistically significant difference). A new balance was reached in hippocampus distinct areas, keeping alive a larger neuronal contingent. There are no evidences in the literature that the decreasing of the apoptosis in CA1 is due to direct action of BETA, but the results suggests that this mortality is a consequence of a previous lesion in DG. In humans, AG use do not cause neurological disabilities after birth [2,3]; however, it is larger the number of
Table 1. Perinatal result for each group.
Discussion
Groups
We analyzed various regions of the brains of BETA- and placebo-treated animals for differences in the number of apoptotic neurons. Our results showed that the groups differed in the number of apoptotic neurons in the CA1 and DG regions of their hippocampus. The administration of BETA
CTR TD 3TD 9TD
Injected animals
Number of fetuses
Number of newborns
Number of newborns/ pregnancy
4 5 4 6
42 48 41 60
41 48 38 2
10.25 9.6 9.5 0.033*
*Fisher’s exact test.
Figure 1. Selected field of control group (CTR) (left), which was treated with the therapeutic dose (TD) of BETA (right). The magenta images indicate apoptotic neurons. The triangles in the vertices represent the boundary of the area and the number of apoptotic cells in the selected field. The yellow scale indicates the distance of 50 mm, which was used as the standard for the calculation of the area.
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M. S. Franc¸a et al.
J Matern Fetal Neonatal Med, Early Online: 1–5
Table 2. Neuronal apoptosis in hippocampus and midbrain. Groups Hippocampus CA1 DG inf. CA1 medial CA2 CA3
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DG sup. Midbrain CTX 12 h CTX 1 h CTX 2 h CTX 3 h CTX 5 h Amygdala
CTR
TD
3TD
p
61.6 (13.8) N¼6 17.6 (4.3) N¼4 45.0 (4.6) N¼3 47.2 (6.5) N¼6 34.6 (10.1) N¼5 45.6 (17.6) N¼4 50 (23–130) N¼5 60 (48–107) N¼5 65 (50–72) N¼4 71 (43–90) N¼4 43 (43–43) N¼1 48 (34–50) N¼3
17.7 (7.1) N¼5 26.3 (4.0) N¼3 42.2 (11.2) N¼4 43.6 (14.3) N¼5 35.0 (19.3) N¼5 25.4 (10.2) N¼5 57 (54–58) N¼3 60 (58–66) N¼3 63 (53–90) N¼4 57 (57–57) N¼1 528 (528–528) N¼1 57 (36–61) N¼3
40.9 (10.2) N¼5 32.2 (8.0) N¼5 39.0 (4.2) N¼4 47.0 (15.8) N¼6 41.7 (9.9) N¼6 37.0 (20.8) N¼4 35 (42–73) N¼6 64 (57–71) N¼2 75 (43–102) N¼5 60 (40–78) N¼3 58 (53–62) N¼2 52 (46–85) N¼6
0.001y 0.011* 0.47* 0.84* 0.63* 0.31* 0.96y 0.91y 0.62y 0.49y 0.25y 0.37y
*Tukey–Kramer. yKruskall–Wallis.
Figure 2. Average ± standard deviation from CA1 by group. (*) indicates significant difference between experimental (TD and 3TD) group and control (CTR) group.
Figure 3. Average ± standard deviation from DG by group. (*) indicates significant? difference between the 3TD group and the control (CTR) group.
psychomotor disturbances involving AG and prematurity [25] or with the pregnancy and stress, as depression [28], attention deficit hyperactivity disorder (ADHD) [25,27,28], schizophrenia [29,30] and epilepsy [31]. There is a positive correlation between the reduction of the encephalon volume of masculine children, ADHD and AG use [27]. The smaller volume of caudate/putamen in human adult with ADHD, described by Castellanos and Acosta [27] can be associated to the decreasing encephalic volume, after AG [1]. It is suggested the volumetric encephalic reduction described by Liggins and Howie [1] and Castellanos and Acosta [27] can be associated with this hippocampus pattern apoptosis and co-made responsible for the genesis of this behavioral alteration [26]. Caudate/ putamen is predominantly responsible for the mental processes related to the prefrontal cortex and very wide connections with hippocampus. It is likely that neuronal cell death contributes to ADHD. The ADHD genesis is correlated with smoking, trauma, stress, alcoholism [28] and AG in premature children [14] or children with stress pregnancy [25]. The opposing effects of BETA on neuronal cell death in DG and CA1 regions can be seen as a result of compensatory mechanisms triggered to minimize the potentially harmful effects of the loss of DG neurons [26], which is likely responsible for the behavioral alterations observed in users of AG. These behavioral changes may reflect lesion in areas such as DG, and of the incapacity of biological reaction of areas as CA1. Were used genetically identical animals, with standard results; however, according to the author’s opinion, in human individuals, with larger genetic and environmental variability, there were different answers after different stimulation. The apoptosis mechanisms could facilitate epileptogenesis in the temporal lobe. In another word, areas with apoptosis increasing, as the DG inf. after AG, can be more susceptible to the propagation of the electric pulse, due to insufficient inhibitions. Together with the findings of earlier studies [24], our results demonstrate that DG responds differently to the absence or excess of glucocorticoids, increased apoptosis in DG and the reduced apoptosis in CA1, demonstrated in this study; and the association between AG and the epileptogenesis of temporal lobe for the decreasing of the epileptic threshold in experimentation animals [31]. The high number of GR in the DG [24] could lead to increased, reducing the neuronal inhibition and facilitating epileptogenesis after appropriate stimulation [31]. In summary, our results showed significant difference in neuronal apoptosis in CA1 and DG regions of the offspring following the administration of BETA in pregnant rats. Such
DOI: 10.3109/14767058.2015.1035639
differences in the neuronal apoptosis between DG and CA1 regions may contribute to the development of ADHD, schizophrenia and epilepsy.
Acknowledgements This work was supported by FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo) Grant No. 04/00877-0.
Declaration of interest The authors declare no conflicts of interests.
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