Effects of postnatal dexamethasone therapy on neurological development in premature infants– from clinical question to animal model

• Main Authors: Cheng-Hsien Tsai, 蔡政憲
• Other Authors: Jeng-Yung Shieh
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/48107344638376111737

碩士 === 國立臺灣大學 === 臨床醫學研究所 === 92 === Dexamethasone (Dex), a synthetic glucocorticoid, is potent and long duration. It decreases early and late manifestations in the inflammatory process and induces cytochrome P450 dependent enzymes. Chronic lung disease (CLD) is an important cause of mortality and morbidity in preterm infants. Because inflammation plays an important role in the pathogenesis of CLD, corticosteroids, in particular Dex, have been widely used to prevent or treat CLD. However, postnatal Dex therapy to prevent CLD was reported to affect somatic growth and neurodevelopmental outcomes followed up at childhood in several clinical studies. We recently reported a group of our preterm infants who had participated in a placebo-controlled, double-blind trial of Dex therapy begun within 12 hours after birth, were more likely to have delays in somatic growth, impaired neuromotor and cognitive dysfunction, and disability at school age. (Appendix 1) Hippocampal formation contains high density of glucocorticoid receptors, is the major inhibitory input of hypothalamic-pituitary-adrenal axis, and has functions of recognition development, hypothalamic control, emotion expression, memory and learning. Our hypothesis is the exposure to postnatal Dex therapy in prematurity may affect the organization of hippocampal formation. The aim of this study was to establish an animal model of developing brain for manipulation, to investigate the effects of a single dose of postnatal Dex on the neuron density and organization of neuroglial cells of hippocampal formation in rat pups. Time-dated Wistar albino pregnant rats were allowed to deliver naturally. Pups were delivered naturally on gestation 21+1 days. The first 24 hour after birth is defined as postnatal day 1 (P1). On P1, pups randomly received either single dose of Dex (Oradexon, 4mg/mL, Organon, Netherlands) or equivalent volume of normal saline intraperitoneally. All the pups were sacrificed on P7 (full-term), P21 (weaning) and P35 (prepubertal period), respectively. Whole brains were removed and embedded in paraffin, then were serially coronally sectioned (7 μm in thickness). Every sixth section was stained with cresyl violet for morphological observation (Nissl-stained). We counted the neuron density using a grid of 200μm x 50μm under 40x10 magnification by a Leitz Aristroplan microscope. Ammon’s horn (CA1) and suprapyramidal blade of middle dentate gyrus (S-DG) at Bregma Plate 31-33 were chosen for comparing the density of neurons between the control and Dex treated pups. The neuron density was counted in triplicate by the profile-based method. Data were analyzed by non-parametric Mann-Whitney test or Kruskal-Wallis test for comparing the differences between means or variance by SPSS statistical software. The results consisted of two parts. In order to study the dose-dependent effect of single pharmacological Dex, every neonate rat in treated groups received Dex 0.5 mg/kg/dose (dose high, Dex-DH) or 0.2 mg/kg/dose (dose low, Dex-DL). 46 Wistar rat pups from 5 litters were analyzed with at least five pups in each group. Their mean body weight were 6.79+0.39 g at P1, 15.21+0.49 g at P7, 57.08+2.18 g at P21, 144.18+17.40 g at P35 in control (NS) group. Their mean brain weight were 0.2991+0.0306 g at P1, 0.6632+0.0462 g at P7, 1.4444+ 0.0383 g at P21, and 1.6360+0.1445 g at P35 in NS group. Both Dex-DL and Dex-DH groups, compared to NS group, had significantly lesser body weight at P7-21-35, and lower brain weight at P7-21, and had dose-dependent effect (p <0.03). But the brain weight at P35 between treated groups and control groups was not significantly different. Morphologically, there were increased pyknotic cells, widening of intercellular space in S-DG and CA1 in Dex-treated groups. Quantitatively, normal neuron density of suprapyramidal-blade dentate gyrus was 83.0+5.3 per 10,000μm2 at P1, peak density at P14-P21(P7, 107.9+3.8 per10,000μm2; P21, 114.7+3.0 per 10,000μm2)，then maintained stable density at P53(127.3+6.0 per10,000μm2). Dex-DL, and Dex-DH had less number of neuron density in S-DG and CA1 at P7-21-35 than control (p <0.02). In addition, the degree of neuronal loss was significantly more prominent in pups received higher dose of Dex (p <0.05). Then, we conducted the second study of immunohistochemical (IHC) staining with frequent intervals to investigate the change of neuroglial cells in the hippocampal formation. After natural delivery, pups randomly received either single dose of Dex (0.5 mg/kg) or equivalent volume of normal saline intraperitoneally. All the pups were sacrificed on P3 (preterm), P7 (full-term), P14 (near-weaning), P21 (weaning), P28 (post-weaning) and P35 (prepubertal period), respectively. Brains were removed, post-fixed and cryoprotected in 30% sucrose/PBS. Segments were freeze-mounted in embedding medium and cryostat sectioning (25 μm). IHC were performed on free floating sections with following monoclonal antibodies. (1) nestin for neuroepithelial stem cell, (2) vimentin for radial-like glial cells, (3) glial fibrillary acidic protein (GFAP) for mature astrocyte, (4) MRC-OX 42 for microglia, (5) Nissl stain for morphological observation. Following staining, five or more sections from each animals were surveyed under low and medium magnification to arrive at scoring based on following scale: baseline (•), mild response (+), moderate response (++), intense response (+++). Criteria for each score were described. In Dex groups, we found the nestin-immunoreactive (IR) signals was inhibited on P3-7, then becoming background staining on P14-21-28-35; vimentin-IR signals was inhibited on P3, then becoming background staining after P7; GFAP-IR astrocyte did not present until P28-P35; MRC-OX 42-IR ramified microglia prematurely presented since P7. By morphological observation, Dex exposure decreased neuron density, inhibited presentation of neuroprecusor cells, delayed mature astrocyte, but prematurely presented ramified microglia. Although only single-dose of pharmacological dose, the effect of Dex on neurological development persisted from preterm—term—weaning—prepubertal periods. Programming effect is defined as the ability of a factor, acting during a defined developmental stage, to exert organizational changes that persist through life. Glucocorticoids probably have programming effects. Exogenous Dex exposure during developing brain may exert inhibiting programming effect on somatic growth, inhibiting neural precursors, decreased presentations of neurons and neuroglials, causing long-term organization change of hippocampal formation. It is implicated with clinical observation that Dex-exposed preterm infants suffered from persisting deleterious outcome at school age. In brief, both clinical observations and our animal study showed postnatal Dex exposure in prematurity have deleterious effects on neurological development, and the effects persisted to school-aged (in clinical study) and prepubertal periods (in animal study). This is probably a programming effect. Key words: brain development, postnatal therapy, hippocampal formation, dexamethasone, programming effect.