American Journal of Obstetrics and Gynecology (2006) 194, 1341–6

www.ajog.org

Lowering the dose of antenatal steroids: The effectsof a single course of betamethasone on somaticgrowth and brain cell proliferation in the rat

Matteo Bruschettini, MD,a,b,c,* Daniel L. A. van den Hove, MSc,a,b Diego Gazzolo, MD,PhD,c,d Harry W. M. Steinbusch, Prof,a Carlos E. Blanco, Profb

Department of Psychiatry and Neuropsychology, Division of Neuroscience, European Graduate School of Neuroscience(EURON),a Department of Pediatrics, Research Institute Growth and Development (GROW),b Faculty of Medicine,University of Maastricht, Maastricht, The Netherlands; G. Gaslini Children’s Hospital, University of Genoa, Genoa,Italyc; Maternal Fetal and Neonatal Health, G Garibaldi Hospital, Catania, Italyd

Received for publication August 5, 2005; revised November 11, 2005; accepted November 28, 2005

KEY WORDSPregnancyPrenatal

corticosteroids

GlucocorticoidsNeurogenesisFisher 344 rat

We investigated the effects of a single course of antenatal betamethasone on neonatal somatic andbrain development. On day 20 of gestation, pregnant rats were injected with either with 170 mgkg�1 body weight of betamethasone (‘‘clinically-equivalent dose,’’ equivalent to 12 mg twice,24 hours apart) or half this dose or vehicle. Pups (8-11 animals per experimental group per time-point per gender) were analyzed at 1 (P1), 2, and 21 days after birth. We report that betameth-asone induced a significant dose-dependent decrease of somatic measurements in both genders.

At P1 cell proliferation was affected by the ‘‘clinically equivalent dose’’ only in the subventricularzone in both genders and in the hippocampus in males. In summary, we show for the first timethat a lower dose (equivalent to 6 mg) induces fewer and less severe effects on somatic growth,

whereas it does not affect cell proliferation within the brain.� 2006 Mosby, Inc. All rights reserved.

Betamethasone is used in the prevention of respira-tory distress syndrome (RDS) in premature infants bornbetween 24 and 34 weeks of gestation. It is known toaccelerate maturation of the fetal lungs1 and reduceneonatal morbidity and mortality.2 However, a singlecourse of antenatal corticosteroids has been shown todecrease birth weight and head circumference at birth3

* Reprint requests: Matteo Bruschettini, MD, Department of Psy-

chiatry and Neuropsychology, Division of Neuroscience, Faculty of

Medicine, Maastricht University, PO Box 616, 6200 MD Maastricht,

The Netherlands.

E-mail: m.bruschettini@np.unimaas.nl

0002-9378/$ - see front matter � 2006 Mosby, Inc. All rights reserved.

doi:10.1016/j.ajog.2005.11.044

and to affect cortisol response to stressors.4 Others,however, found no growth restriction after antenatalglucocorticoids.5

In animal models, antenatal glucocorticoids havebeen shown to cause a wide range of side effects, eg,reduction of cerebellar DNA content6 and loss of synap-tic density in the frontal neocortex, caudate putamen,and hippocampus.7 In many of these animal studiesthe doses used had been higher than in the human situ-ation. Recently, our group showed that a single courseof antenatal betamethasone (equivalent to 2 injections,12 hours apart, in the clinic) impaired growth and cellproliferation within the brain of the rat.8 In this study,

1342 Bruschettini et al

our primary aim was to investigate the effects of both astandard, clinically equivalent dose (CD) of antenatalbetamethasone (equivalent to 2 injections 24 hoursapart) and of half this dose (HD), focusing on somaticgrowth and neonatal brain development.

Methods

The animal studies described here were all approved bythe Animal Ethics Board of the University of Maas-tricht, The Netherlands.

Pregnant Fisher 344 dams (Charles River, Maas-tricht, The Netherlands; pregnancy confirmed by vagi-nal plug) were delivered to our animal facility on day 14of gestation (G14). The animals were kept under stan-dard laboratory conditions with 12 hours light/12 hoursdark and standard rat chow and water ad libitum. Therats were randomly assigned to an experimental grouptreated either with a clinically equivalent dose (CD) ofantenatal betamethasone, half this dose (HD), or vehicle(as specified in the next paragraph). Pups were exam-ined at P(postnatal day)1, P2, and P21. For the agebetween P2 and P21, the pups were all cross-fostered todams that had given birth on the same day and hadreceived vehicle-only treatment (to prevent a possiblebetamethasone effect on maternal behavior influencingthe results). All the litters were kept at 8 pups per damand the pups were kept with the mother until P21, ie,they were not weaned.

Betamethasone treatment

As mentioned previously, women threatening to deliverpreterm are administered 12 mg betamethasone (Celes-tone Chronodose, Schering-Plough, Utrecht, TheNetherlands) twice 24 hours, apart. With an averageweight of around 80 kg, this corresponds to 170 mg kg�1

betamethasone with a plasma half-life of 6 hours in thehuman. The second injection therefore occurs at an inter-val of 4 half-lives. Within the rat, betamethasone has aplasma half-life of 2 hours.9 An equivalent dose for a ratwould then be 2 doses of 170 mg kg�1, 8 hours apart.

Betamethasone was diluted in its own buffer to aconcentration of 230 mg mL�1. The animals were in-jected in the nape of the neck with a clinically equivalentdose (CD) of antenatal betamethasone (170 mg kg�1,corresponding to 12 mg in the clinic), half this dose(HD; 85 mg kg�1, corresponding to 6 mg in the clinic)or vehicle-only at 9 AM and 5 PM on G20. All theanimals delivered on G22, which was designated aspup age P0. For birth measurements (within 90 minutesafter delivery) the pups were sexed, weighed, and hadtheir crown-tail (C-T) length and head diameter mea-sured by using a digital vernier caliper. All measure-ments were taken by the same investigator (M.B.,blind to the experimental group) to preserve consistency.

During this time the dam was never left without anypups and the pups were not separated from their damfor more than 3 minutes to minimize the stress levelsexperienced.

Tracer method

We used the 3H-thymidine (3H-Thy) incorporationmethod to calculate mitotic activity in specific brain re-gions at 1, 2, and 21 days after birth by measuring theDNA synthetic rate, which is proportional to the rateof cell proliferation.

On their assigned day (P1, P2, or P21) the pups wereinjected with 5 mCi (g body weight)�1 3H-Thy (25 Cimmol�1, 1 m Ci mL�1 in 0.9 % NaCl, Amersham Phar-macia Biotech, Roosendaal, The Netherlands) by sub-cutaneous injection into the nape of the neck. After3H-Thy infusion the pups were kept at 34(C and 75%humidity in a pediatric incubator. Exactly 1 hour laterthe pups were killed by decapitation and the brainswere quickly removed and dissected. We isolated 4 brainregions: the olfactory bulbs (OB), the cerebellum, theentire hippocampal formation, and the subventricularzone (SVZ) contained within the rostral forebrain. Themicrodissected regions were then weighed, quicklysnap frozen in liquid nitrogen, and stored at �70(C.Subsequently, the heart, lungs, liver, and kidneys weretaken and weighed. All dissections were performed bythe same investigator (M.B.) to preserve consistency.

The dissected brain regions were then placed in 350 mL(olfactory bulbs and hippocampus) or 1 mL (cerebellumand SVZ) precooled lysis buffer containing: 137 mmol/LNaCl, 20 mmol/L Tris-HCl (pH 8.0), 1% NP-40, 10%glycerol, and a complete protease inhibitor tablet (Roche,Woerden, The Netherlands). The samples were thenhomogenized using a Bead Beater (Biospec Products,Bartlesville, OK) for 3 ! 30 seconds, each time withcooling of the samples on ice between runs.

An aliquot of the homogenate was used to extract allcellular DNA using a standard trichloracetic acid pre-cipitation protocol to determine the amount of 3H-Thythat was taken up by proliferating cells and incorpo-rated into newly synthesized DNA during the 1 hour ex-posure. Essentially, this measure represents the productof the number of S phase cells within the sample timesthe DNA synthetic rate of these S phase cells. However,the incorporation of 3H-Thy into DNA depends on theamount of precursor taken up by the tissue. As a conse-quence, differences in, for example, blood flow betweenexperimental groups might result in differences in 3H-Thy incorporation, which does not reflect differencesin mitotic activity. For this purpose, another aliquotof the homogenate was used to measure total amountof radioactivity in the tissue fraction.

Homogenized tissue and DNA samples were solubi-lized in 1 mL Soluene-350 (Packard Instruments,

Bruschettini et al 1343

Groningen, The Netherlands) at 50(C for 3 to 24 hoursor until the samples were completely dissolved. After-ward 5 mL of Hionic-Flour scintillation cocktail(Packard Instruments) was added and the sampleswere read for 20 minutes on a Wallac WinSpectral1414 liquid scintillation counter (PerkinElmer Life Sci-ences, Los Alamos, NM). The appropriate quenchcurves were produced by using tritium standards addedto homogenized brain tissue and subsequently used toconvert the sample counts per minute to disintegrationsper min (dpm). The dpm measures were corrected forthe wet weight of tissue (mg) and the percentage of 3H-Thy incorporated into DNA relative to the total amountof radioactivity counted in the appropriate tissue of eachbrain region was calculated.

Statistics

We used a standardized and randomized block designfor these studies whereby each dam had 1 pup of eachsex used at each time point to remove any litter effects.Differences in litter size were tested using a 1-wayanalysis of variance (ANOVA). Mortality was testedusing the Fisher exact test. In all other cases, the datawere evaluated with a 3-way ANOVA (experimentalgroup ! gender ! age). Effects were analyzed in moredetail with least significant difference (LSD) post hoctests (P ! .05). All statistics were carried out using SPSSsoftware version 11.5 (SPSS Inc, Chicago, IL). Data arepresented as means G SEM.

Results

Litter size and preweaning mortality

No significant differences between groups were observedin litter size (7.6, 7.3, and 9.3 pups/litter for vehicle, HD,and CD, respectively), and preweaning mortality (6 pupsdied: 2 vehicle and 4 CD).

Body weight, head diameter and C-T lengthat birth

Somatic measurements at birth (P0) are shown inFigure 1. Antenatal betamethasone caused a significantdose-dependent body weight reduction in the CD(�8.0%) and HD (�3.1%) groups. The same effectwas found for C-T length (CD: �3.6%, HD: �1.6%).

CD decreased head diameter at birth compared withboth vehicle (�3.7%) and HD, whereas head diameterwas not significantly reduced by HD. There was atendency toward a significant effect of HD on headdiameter in males (P = .098).

Female pups were smaller compared with males onall parameters (body weight and C-T length: P ! .01;head diameter P ! .05).

Postnatal somatic growth and relativeorgan weights

Somatic measures were affected in the treated pupsonly on P1 (Table), with a gender-independent reductionin body weight (P ! .01) and in C-T length (P = .041),and a tendency for head diameter (P = .083). Post hocanalysis showed that HD did not cause growth retarda-tion, but CD did (body weight: P = .028; C-T length:P = .011; head diameter: P = .032). C-T length mea-sures were not taken at P21 and gender by experimen-tal group interactions were not found for any somaticmeasure at any time point.

Overall, relative weights of both the liver and lungs(% of body weight) were reduced by the betamethasonetreatment, whereas heart and kidneys were not affected(data not shown).

Brain and brain region weights

CD betamethasone treatment decreased brain weight inboth male (�8.7%, P = .039) and female (�6.2%, P =.026) offspring at P1, but not relative weight (brain/bodyweight ratio).

CD caused an overall decrease in both cerebellum(�11.2%, P ! .01) and hippocampus (�5.4%, P= .041)weight compared with the HD. At P1, SVZ weight wasdecreased by both doses compared with vehicle inthe males (CD: �21.7%, P ! .01; HD: �15.1%,P = .016). At P2, cerebellar weight was reduced onlyby HD (�17.4%, P = .037), whereas hippocampalweight by CD (�22.3%, P = .021) in the female.

Cell proliferation within the brain

At P1, cell proliferation within the hippocampuswas higher in CD than in HD in the males (C64.2%,

Figure 1 Overview of birth measures. Values are expressed aspercentage of the vehicle group and represent Mean C SEM,n = 20 to 34/experimental group/gender. *P ! .05 compared

with Vehicle group; **P ! .001 compared with Vehicle group;#P ! .05 compared with HD; ##P ! .001 compared with HD;LSD test.

1344 Bruschettini et al

Table Betamethasone effect on somatic growth

Gender and dose Parameter P1 P2 P21

Male Vehicle Body weight (g) 5.2 G 0.1 5.8 G 0.2 26.1 G 0.6HD 4.8 G 0.1 5.3 G 0.1 25.8 G 0.4CD 4.6 G 0.1* 5.5 G 0.2 24.6 G 0.6

Female Vehicle 4.9 G 0.1 5.4 G 0.1 24.6 G 1.1HD 4.7 G 0.2 5.2 G 0.2 24.3 G 0.4CD 4.5 G 0.1y 5.0 G 0.2 25.0 G 0.9

Dose effect P = .004 NSGender effect P = .059 P = .04Male Vehicle Head diameter (mm) 10.4 G 0.2 11.1 G 0.3 15.6 G 0.3

HD 10.2 G 0.2 10.4 G 0.2 15.6 G 0.2CD 10.1 G 0.2 10.6 G 0.3 15.3 G 0.2

Female Vehicle 10.3 G 0.1 10.6 G 0.3 15.4 G 0.3HD 10.1 G 0.3 10.3 G 0.2 15.2 G 0.2CD 10.3 G 0.2 10.2 G 0.3 15.3 G 0.3

Dose effect P = .04 NSGender effect NS NSMale Vehicle C–T length (mm) 44.7 G 0.6 47.6 G 0.7

HD 43.9 G 0.5 46.0 G 0.5CD 42.3 G 0.5y 46.9 G 1.0

Female Vehicle 43.9 G 0.5 46.5 G 0.6HD 43.0 G 0.6 46.3 G0.6CD 42.9 G 1.0 44.8 G 1.0

Dose effect NS NSGender effect NS NS

Effect of antenatal betamethasone on somatic growth parameters, (mean G SEM, n = 8 to 11/gender/experimental group/timepoint). Data were

evaluated with a 3-way ANOVA (experimental group ! gender ! age). Symbols used for post hoc differences.

* P ! .01 compared with vehicle.y P ! .05 compared with vehicle.

P= .028) (Figure 2). In the SVZ, the CD group showed ahigher degree of cell proliferation compared with vehicle(C55.4%, P = .007) and in males compared with bothHD (C82.8%, P = .011) and vehicle (C71.2%, P =.019). In the rest of the brain, cell proliferation was higherin CD compared with both HD (C38.2%, P= .002) andvehicle (C34.3%, P = .002).

At P2, cell proliferation was not significantly changedby betamethasone. At P21, CD increased cell prolifer-ation within the hippocampus compared with vehicle inthe females (C4.3%, P = .050) (data not shown). In thecerebellum, CD increased cell proliferation comparedwith vehicle (C12.5%, P = .012) and HD (malesonly; C11.0%, P = .014).

Cell proliferation was lower in males in OB (�6.2%,P = .039), hippocampus (�16.1%, P ! .01), and SVZ(�12.9%, P ! .01).

Comment

This study shows that a lower dose of antenatal beta-methasone results in less severe effects on somaticgrowth while it does not seem to impair cell proliferationwithin the neonatal brain.

Recently, our group showed that a single course ofbetamethasone consisting of 2 injections 4 hours apartin the rat, corresponding to 2 injections 12 hours apartin the human, induced somatic growth retardation and areduction of brain cell proliferation.8 In the currentstudy we chose an 8-hour interval, because the Consen-sus Statement2 recommends a 24-hour interval betweenthe 2 injections. In addition, we used lower doses: a clin-ically equivalent dose (CD) to attempt to replicate theclinical situation of 12 mg twice, 24 hours apart; andhalf this dose (HD). This latter dose regimen (6 mgtwice, 24 hours apart) has been recently suggested,because it may be an equally effective but less toxictreatment.10

A major concern with regard to the interpretation ofanimal studies examining the effects of glucocorticoidson neural measures is the comparison of the stage ofbrain development. Estimates of the rat equivalent ageof a term human in respect of neural development haveranged from 7 to 24 days of postnatal age with a generalconsensus that a 10- to 14-day-old rat is equivalent to aterm human.11 Term rats are therefore equivalent tovery preterm human infants, ie, those who would receiveantenatal glucocorticoids in utero. A further concern isthe comparison of neural cell cycle times. Ideally, we

Bruschettini et al 1345

would expose the rat brain cells to betamethasone forthe same number of cell cycles as occurs in the human.Fortuitously, the cell cycle time of the developing pri-mate (and presumably human) brain is 3 to 5 times lon-ger than that of a rodent fetus in the third trimester ofpregnancy, which is similar to the difference in beta-methasone half-life.12

Somatic growth

The effects of glucocorticoids in pregnancy on growthrestriction are not clear in human studies.3,5 We showthat a single course of antenatal betamethasone retardssomatic growth. This might be the result of a reductionof circulating levels of growth factors such as growth hor-mone (GH) and therefore insulin-like growth factor1 (IGF-1), which are known to stimulate growth.13 Inter-estingly, at birthHDaffected bodyweight andC-T lengthless than CD. Moreover, head diameter was not de-creased by HD at birth. Of note, the fast growth catch-up observed after betamethasone treatment does notnecessarily imply a better outcome. On the contrary, areduced birth size, followed by a fast weight gain, hasbeen associated with an additional risk of disease in laterlife.14

In our previous investigation there was no differencein the degree of growth retardation caused by the 2doses (except for C-T length in males).8 This discrep-ancy may be due to the lower dose now been used,revealing the presence of a ceiling effect in terms ofgrowth retardation. The longer interval between the 2injections may be an alternative explanation, resultingin a lower peak concentration of betamethasone inboth the dam and fetus. Obviously, a combination of

Figure 2 Brain cell proliferation at P1. Graph showing the

effect of antenatal betamethasone on postnatal brain cell pro-liferation (expressed as % 3H-Thy incorporation; see text) inmales and females. CD betamethasone treatment caused sig-

nificant increases in proliferation in HIP and SVZ at P1,whereas HD did not affect cell proliferation in any of the brainregions. For comparison, brain cell proliferation levels at P21

in the vehicle group are depicted by dots. Values representMean C SEM, n= 11 to 17/experimental group/time. *P !.05 compared with Vehicle; #P ! .05 compared with HD.CER, Cerebellum; HIP, hippocampus; Rest, rest of the brain.

these 2 factors, ie, lower dose and longer injection inter-val, is also a possible explanation. In line with this find-ing, HD does not produce a reduction in head diameter,as observed in human neonates after a single course ofbetamethasone.3 Of note, a reduced head diameter atbirth has been associated with learning problems inschool-age children.15

The milder effects found in the current study werealso visible in the postnatal growth of the pups, char-acterized by a prompt catch-up, with a reduction inweight and C-T length only by CD at P1, and nodifferences after P1. However, a rapid postnatal weightgain may increase the incidence of coronary heartdisease in adult life.14

Taken together, these findings suggest that loweringthe dose of antenatal betamethasone may result in fewerand less severe effects on somatic growth.

Cell proliferation within the neonatal brain

It is known that glucocorticoids are essential for mat-uration in the developing central nervous system (CNS),where they play a pivotal role in the remodeling of axonsand dendrites, and in cell survival.16 Antenatal glucocor-ticoids administration, however, has been shown to beable to suppress the proliferation of cells derived fromthe hippocampus of rat embryos,17 possibly perma-nently altering brain structure.18

In the current study, the effects of antenatal beta-methasone on cell proliferation within the brain areless clear compared with those on somatic growth.Whereas HD did not affect cell proliferation, CDincreased cell proliferation in the hippocampus (malesonly) and SVZ (both genders) at P1. This enhancedcell proliferation might be the rebound of an inhibi-tion of cell proliferation before this time point, asobserved in our previous investigation, which showeda catch-up in cell proliferation at P2 in all the brainregions studied after an initial decrease observed atP1.8 The timing of events, ie, the inhibition of cellproliferation, followed by a catch-up, might haveshifted, with the inhibition now occurring before P1because of the longer injection intervaldie, lower con-centration of betamethasone (previously described)dused in the current study.

The altered proliferation rate that we report mightinvolve a downregulation in the expression of neuro-trophic factors, such as brain-derived neurotrophicfactor (BDNF) and S100B protein. BDNF is implicatedin neuronal proliferation, migration, and differentiation,whereas S100B protein promotes proliferation in bothneuronal and glial cells.19,20 Interestingly, these growthfactors have been shown to be reduced by antenatal glu-cocorticoids in the rat hippocampus21 and in the urineof human infants.22 In particular, S100B protein isreduced in the hippocampus in males only,23 possibly

1346 Bruschettini et al

reflecting different patterns of brain development in the2 sexes.24

As described previously regarding somatic growth,the current data suggest also that cell proliferationwithin the brain is spared by halving the dose ofantenatal betamethasone.

Conclusion

We show that a single course of antenatal betametha-sone (equivalent to 2 injections, 24 hours apart) inducessomatic growth retardation and affects cell proliferationwithin the hippocampus and SVZ. Moreover, we showfor the first time that a lower dose (equivalent to 6 mgtwice, 24 hours apart) induces fewer and less severeeffects on somatic growth, whereas it does not affect cellproliferation within the brain. This suggests that lower-ing the dose of antenatal betamethasone might indeedbe effective in minimizing related side effects, onceclinical trials show its effectiveness for inducing lungmaturation. We therefore reiterate the need for random-ized clinical trials with a low-dose regimen.

References

1. Ballard PL, Ballard RA. Scientific basis and therapeutic regimens

for use of antenatal glucocorticoids. Am J Obstet Gynecol 1995;

173:254-62.

2. Antenatal corticosteroids revisited: repeat courses. NIH Consensus

Statement 2000;17:1-18.

3. Thorp JA, Jones PG, Knox E, Clark RH. Does antenatal cortico-

steroid therapy affect birth weight and head circumference? Obstet

Gynecol 2002;99:101-8.

4. Davis EP, Townsend EL, Gunnar MR, Georgieff MK, Guiang SF,

Ciffuentes RF, et al. Effects of prenatal betamethasone exposure

on regulation of stress physiology in healthy premature infants.

Psychoneuroendocrinology 2004;29:1028-36.

5. Vermillion ST, Soper DE, Chasedunn-Roark J. Neonatal sepsis af-

ter betamethasone administration to patients with preterm prema-

ture rupture of membranes. Am J Obstet Gynecol 1999;181:320-7.

6. Velazquez PN, Romano MC. Corticosterone therapy during gesta-

tion: effects on the development of rat cerebellum. Int J Dev Neu-

rosci 1987;5:189-94.

7. Colberg C, Antonow-Schlorke I, Muller T, Schubert H, Witte OW,

Schwab M. Recovery of glucocorticoid-related loss of synaptic

density in the fetal sheep brain at 0.75 of gestation. Neurosci

Lett 2004;364:130-4.

8. Scheepens A, van de Waarenburg M, van den Hove D, Blanco CE.

A single course of prenatal betamethasone in the rat alters

postnatal brain cell proliferation but not apoptosis. J Physiol

2003;552:163-75.

9. Tamvakopoulos CS, Neugebauer JM, Donnelly M, Griffin PR.

Analysis of betamethasone in rat plasma using automated solid-

phase extraction coupled with liquid chromatography-tandem

mass spectrometry: determination of plasma concentrations in

rat following oral and intravenous administration. J Chromatogr

B Analyt Technol Biomed Life Sci 2002;776:161-8.

10. Jobe AH, Soll RF. Choice and dose of corticosteroid for antenatal

treatments. Am J Obstet Gynecol 2004;190:878-81.

11. Clancy B, Darlington RB, Finlay BL. Translating develop-

mental time across mammalian species. Neuroscience 2001;105:

7-17.

12. Kornack DR, Rakic P. Changes in cell-cycle kinetics during the

development and evolution of primate neocortex. Proc Natl Acad

Sci U S A 1998;95:1242-6.

13. Price WA, Stiles AD, Moats-Staats BM, D’Ercole AJ.

Gene expression of insulin-like growth factors (IGFs), the type 1

IGF receptor, and IGF-binding proteins in dexamethasone-

induced fetal growth retardation. Endocrinology 1992;130:

1424-32.

14. Gluckman PD, Hanson MA. Living with the past: evolution,

development, and patterns of disease. Science 2004;305:1733-6.

15. Stathis SL, O’Callaghan M, Harvey J, Rogers Y. Head circumfer-

ence in ELBW babies is associated with learning difficulties and

cognition but not ADHD in the school-aged child. Dev Med Child

Neurol 1999;41:375-80.

16. Meyer JS. Early adrenalectomy stimulates subsequent growth and

development of the rat brain. Exp Neurol 1983;82:432-46.

17. Yu IT, Lee SH, Lee YS, Son H. Differential effects of corticoster-

one and dexamethasone on hippocampal neurogenesis in vitro.

Biochem Biophys Res Commun 2004;317:484-90.

18. Matthews SG. Antenatal glucocorticoids and programming of the

developing CNS. Pediatr Res 2000;47:291-300.

19. Arcuri C, Bianchi R, Brozzi F, Donato R. S100B increases prolif-

eration in PC12 neuronal cells and reduces their responsiveness to

nerve growth factor via Akt activation. J Biol Chem 2005;280:

4402-14.

20. Michetti F, Gazzolo D. S100B protein in biological fluids: a tool

for perinatal medicine. Clin Chem 2002;48:2097-104.

21. Schaaf MJ, Hoetelmans RW, de Kloet ER, Vreugdenhil E. Corti-

costerone regulates expression of BDNF and trkB but not NT-3

and trkC mRNA in the rat hippocampus. J Neurosci Res 1997;

48:334-41.

22. Gazzolo D, Kornacka M, Bruschettini M, Lituania M, Giovannini

L, Serra G, et al. Maternal glucocorticoid supplementation and

S100B protein concentrations in cord blood and urine of preterm

infants. Clin Chem 2003;49:1215-8.

23. Bruschettini M, Van den Hove DLA, Gazzolo D, Bruschettini P,

Blanco CE, Steinbusch HWM. A single course of antenatal beta-

methasone reduces neurotrophic factor S100B concentration in

the hippocampus and serum in the neonatal rat. Brain Res Dev

Brain Res 2005;159:113-8.

24. MacLusky NJ, Naftolin F. Sexual differentiation of the central

nervous system. Science 1981;211:1294-302.