Magnesium sulfate ameliorates maternal and fetal inflammation in a rat model of maternal infection

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Research www.AJOG.org

BASIC SCIENCE: OBSTETRICS

Magnesium sulfate ameliorates maternal and fetalinflammation in a rat model of maternal infectionHima B. Tam Tam, MD; Oonagh Dowling, PhD; Xiangying Xue, MD;Dawnette Lewis, MD, MPH; Burton Rochelson, MD; Christine N. Metz, PhD

OBJECTIVE: Magnesium sulfate is proposed to have neuroprotectiveeffects in the offspring. We examined the effects of maternal magne-sium sulfate administration on maternal and fetal inflammatory re-sponses in a rat model of maternal infection.

STUDY DESIGN: Pregnant rats were injected with saline, Gram-negativebacterial endotoxin lipopolysaccharide or lipopolysaccharide with magne-sium sulfate (pre- and/or after lipopolysaccharide) to mimic infection. Ma-ternal blood, amniotic fluid, fetal blood, and fetal brains were collected 4hours after lipopolysaccharide and assayed for tumor necrosis factor, inter-leukin-6, monocyte chemoattractant protein-1, and growth-related onco-gene-KC. In addition, the effect of magnesium sulfate on cytokine produc-
tion by an astrocytoma cell line was assessed.
Am J Obstet Gynecol 2011;204:364.e1-8.

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glandin cascade leadindoi: 10.1016/j.ajog.2010.11.006

RESULTS: Lipopolysaccharide administration induced tumor necrosisfactor, interleukin-6, monocyte chemoattractant protein-1, andgrowth-related oncogene-KC expression in maternal and fetal compart-ments. Maternal magnesium sulfate treatment significantly attenuatedlipopolysaccharide-induced multiple proinflammatory mediator levelsin maternal and fetal compartments.

CONCLUSION: Antenatal magnesium sulfate administration significantlyameliorated maternal, fetal, and gestational tissue-associated inflamma-tory responses in an experimental model of maternal infection.

Key words: chemokines, cytokines, fetal brain damage, GRO-KC,magnesium sulfate, maternal infection, MCP-1, neuroprotection,
preterm labor
Cite this article as: Tam Tam HB, Dowling O, Xue X, et al. Magnesium sulfate ameliorates maternal and fetal inflammation in a rat model of maternal infection.

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Preterm birth is a major cause of peri-natal morbidity and accounts for

approximately 70% of all perinatal mor-tality.1 Maternal infection, a major riskactor for preterm labor,1,2 is associated

with significant neonatal morbidity, in-

From the Division of Maternal Fetal Medicine,Department of Obstetrics and Gynecology(Drs Tam Tam, Lewis, Rochelson); and TheCenter for Immunology and Inflammationand Merinoff Center for Patient-OrientedResearch (Drs Dowling, Xue, Metz), TheFeinstein Institute for Medical Research, NorthShore-LIJ Health System, Manhasset, NY.

Presented in abstract form and as an oralpresentation at the Annual Society of Maternal-Fetal Medicine Meeting, Feb. 6, 2010.

Received Aug. 23, 2010; accepted Nov. 2,2010.

Reprints not available from the authors.

The Oxenhorn Family and the FeinsteinInstitute for Medical Research providedfinancial support.

Dr Dawnette Lewis is currently at Long IslandCollege Hospital, Brooklyn, NY.

0002-9378/$36.00© 2011 Mosby, Inc. All rights reserved.

cluding neurologic injury.3-5 The mater-nal inflammatory response to infectionis characterized by the production ofproinflammatory mediators such as cy-tokines and chemokines, which can leadto tissue injury when produced in excess.

Lipopolysaccharide (LPS), the Gramnegative bacterial endotoxin, has beenused in laboratory animals to character-ize the underlying pathogenesis of in-flammation/infection-mediated pre-term labor,6 to investigate infection-ssociated fetal neuronal damage,7-10

and to test various potential therapeuticinterventions.11-13 Endotoxin adminis-tration to pregnant laboratory animalsinduces proinflammatory mediator pro-duction in the maternal and fetal com-partments. This model mimics the ele-vated levels of cytokines and chemokines(eg, tumor necrosis factor-alpha [TNF],interleukin-6 [IL-6], monocyte che-moattractant protein [MCP-1, CCL2])observed in maternal and fetal compart-ments during infection and/or pretermlaboring humans.14-16 Excessive and sus-tained inflammatory mediator produc-tion is proposed to activate the prosta-

g to preterm birth, f

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a risk factor for neurologic damage.5,17,18

In addition, fetal exposure to inflamma-tory mediators is understood to belinked to neurologic insults in the off-spring, including long-term neurocogni-tive dysfunction and cerebral palsy.5,17,18

Several reports support the neuro-protective effect of magnesium sulfate(MgSO4) in the fetus. A randomizedclinical trial by Rouse and colleagues19

showed a significantly lower rate of cere-bral palsy among infants born to womenat 24-31 weeks’ gestation who receivedMgSO4 tocolysis for preterm labor. A re-ent metaanalysis20 and the Cochrane

Review21 also supported this association.Although MgSO4 has been used in ob-

tetrics for suppressing preterm labor, itsxact mechanism of action is unknown.esearch in our laboratory revealing thatgSO4 attenuated cytokine produc-

ion by cultured endothelial cells high-ights the antiinflammatory activity of

gSO4.22 Additional in vivo studiesemonstrating exaggerated inflamma-ory responses in Mg-deficient animalsn the absence23 and presence24 of LPSurther support the antiinflammatory ef-
ects of MgSO4.
an Journal of Obstetrics & Gynecology 364.e1

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Given the emerging evidence for (1) theassociation between neuronal injury andcytokine-mediated damage in the fetus,(2) the potential protective role of antena-tal MgSO4 therapy on the development oferebral palsy, and (3) the antiinflamma-ory activity of MgSO4, we sought to inves-igate the effects of MgSO4 on cytokine and

chemokine concentrations within mater-nal and fetal circulation, as well as in theamniotic fluid and fetal brain using a ro-dent model of LPS-induced maternal sys-temic infection, and to examine the effectsof MgSO4 on inflammatory mediator pro-

uction by a human neuronal cell line.

MATERIALS AND METHODSWe obtained approval from the Institu-tional Animal Care and Use Committee(IACUC) before conducting our study.Timed pregnant Sprague-Dawley rats(E9 and E11) (Charles River, Wilming-ton, MA) were housed individually un-

FIGURE 1MgSO4 treatment suppresses cytokconcentrations in maternal serum

A, TNF, B, IL-6, C, MCP-1, and D, GRO-KC conceinutes after saline or LPS injection (in the presen

s individual data points (●) with geometric meaGRO-KC, growth-related oncogene-KC; IL-6, interleukin-6; LPS, lip

agnesium sulfate; MLM, MgSO4-LPS-MgSO4; MLS, MgSO4-LPSaline-saline. � and �� indicate P � .05 and P � .01, respec

Tam Tam. Antenatal MgSO4 ameliorates inflammation. Am

der normal environmental conditions (

364.e2 American Journal of Obstetrics & Gynecolo

with free access to standard rat chow andwater for at least 3 days before experi-mentation. LPS (strain Escherichia coli055:B5) was purchased from Sigma-Al-drich (St. Louis, MO). MgSO4 was pur-hased from APP Pharmaceuticals, LLCSchaumburg, IL). Pregnant rats (gesta-ional day 19) were randomly assignedy a technician blinded to the study de-ign into 1 of 5 treatment groups (n � 6er group): (1) saline (given subcutane-usly [sc] q20 minutes for a period of 4ours before and a period of 4 hours af-er saline intraperitoneally [ip]) (SSS)-egative controls; (2) LPS (1 mg/kg, ip)nd saline (sc q20 minutes for a period ofhours before and a period of 4 hours

fter LPS) (SLS) to mimic infection-as-ociated preterm labor; (3) LPS (1 mg/g, ip) and MgSO4 (270 mg/kg load andhen 27 mg/kg sc q20 minutes for a pe-iod of 4 hours before and for a period of

hours after LPS) (MLM); (4) LPS

e and chemokinemin post-LPS

ations were measured in the maternal serum 90r absence of MgSO4 treatment). Data are shown�).ysaccharide; MCP-1, monocyte chemoattractant protein; MgSO4,e; SLM, saline-LPS-MgSO4; SLS, saline-LPS-saline; SSS, saline-, comparing SSS to SLS or SLS to MLM.

bstet Gynecol 2011.

1 mg/kg, ip) and MgSO4 (270 mg/kg %

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oad and then 27 mg/kg sc q20 minutes 4ours before LPS only and saline given sc20 minutes 4 hours after LPS) (MLS);r (5) LPS (1 mg/kg, ip) and saline givenc q20 minutes for a period of 4 hoursefore LPS and MgSO4 (270 mg/kg load

and then 27 mg/kg sc q20 minutes) for 4hours after LPS (SLM). A similar MgSO4

regimen was used by Hallak et al25 andthe LPS administration protocol (route,dose, timing) was similar to Kumral etal.26 Maternal blood was drawn (retroor-bitally) 1½ hours after ip administrationof LPS or saline. Four hours post-LPS (orsaline) injection, the dams were eutha-nized by CO2 (1 group of rats were eu-hanized 90 minutes post-LPS for fetalrain messenger RNA [mRNA] studies).aternal blood was collected via cardiac

uncture, and amniotic fluid was col-ected from each gestational sac. Fetallood (pooled from the pups of eacham) and brains were obtained at theime of decapitation. Blood specimensere collected in nonheparinized tubes,

he blood was allowed to clot for 30 min-tes, and then specimens were centri-

uged to collect serum. Fetal brains wereash frozen in liquid nitrogen, and bothrain and serum samples were stored at80° C.Magnesium (Mg) concentrations ofaternal blood and amniotic fluid were

ssessed by the North Shore-LIJ Healthystem Core Laboratories. For cytokinessessment, fetal rat brains were homog-nized in 4 volumes of phosphate-buff-red saline (PBS) containing 0.1%P-40 and protease inhibitors. Cyto-

ines/chemokines (TNF, IL-6, MCP-1,nd growth-related oncogene-KC [GRO-C, CXCL1]) in the rat serum, amnioticuid, and brain homogenates were ana-

yzed using Luminex XMAP technologyMillipore, St. Louis, MO). Fetal brain cy-okine/chemokine concentrations weredjusted for protein concentrations (Bio-ad protein method, Hercules, CA). Cyto-ine data are shown as individual dataoints with geometric means. The sensitiv-

ties (pg/mL) of the assays were as follows:NF: 4.88-24.4; IL-6: 9.8-24.4; MCP-1:.81-4.88; and GRO-KC: 2.06-18.44nd the precisions (intraassay/interassay,

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www.AJOG.org Basic Science: Obstetrics Research

IL-6: 10.37/14.3; MCP-1: 3.81/16.5; andGRO-KC: 7.39/14.1.

The RNA was isolated from frozen fetalbrains (at 90 minutes and 4 hours post-LPS) using the RNeasy kit (Qiagen, Valen-cia, CA) and RNA preparations with A260:280 ratios �1.9 were analyzed. The relativexpression of GRO-KC, MCP-1, andXCR2 mRNAs were assessed by quanti-

ative real-time polymerase chain reactionQ-PCR) using Roche Universal Probe Li-rary technology. Reactions (performed inuplicate) were completed using 200 ngNA, Eurogentec onestep qRT-PCR mas-

er mix, and 7900HT Fast Real-Time PCRystem (Applied Biosystems, Foster City,A). Conditions used for the RT-PCR re-ction were: 48° C (30 minutes) and 95° C10 minutes), then 45 cycles of 95° C (15econds) and 60° C (1 minute) using spe-ific primer sequences: GRO-KC: F-acactccaacagagcacca, R-tgacagcgcagct-attg; MCP-1: F-agcatccacgtgctgtctc-gatcatcttgccagtgaatgagt; CXCR-2: F-tctttgctgtggtcctcgt, R-tgaacaggacaatgttg-aggg. Relative gene expression levels werealculated using the ��Ct method and ex-ression levels were normalized to theousekeeping gene GAPDH (Universalrobe Library Rat GAPDH Gene Assay,oche). Data are presented as mean foldhange over saline control (mean � SEM).

To investigate the effects of MgSO4 onrain cytokine responses ex vivo, the87-MG human neuronal gliobalstoma

strocytoma cell line (ATCC, Manassas,A) was cultured in 96-well plates inMEM containing 10% fetal bovine se-

um and penicillin-streptomycin, andhen treated with vehicle or MgSO4

(0.1-25 mM) for 3 hours before TNFtreatment (50 ng/mL) or IL-1� (1 ng/

L) (n �4 wells per condition). After anvernight incubation, cell-free cultureupernatants were collected and assayedor MCP-1 and IL-8 by ELISA (R&D Sys-ems, Minneapolis, MN). All assays wereepeated 2 times and data are shown asean (� SEM).

Statistical analysesThe rat cytokine data (normalized by alog transformation) was analyzed in 2steps. First, 2-sample t tests, using a logransformation of the cytokine levels,
ere used to compare the SSS (negative t
ontrol) group with the SLS (positiveontrol) group to confirm induction byPS (P � .05). In step 2, separate 1-waynalysis of variances (ANOVAs) weresed to analyze the cytokines concentra-

ions in the maternal and fetal compart-ents. On significant ANOVA findings

P � .05), each group (MLS, SLM, andLM) was compared with the positive

ontrol group (SLS) using the Dunnett’sest. The standard assumptions of Gauss-an residuals and equality of variance wereested. Q-PCR data were analyzed with-way ANOVAs with significance set at� .05.

RESULTSAt 90 minutes after maternal LPS admin-istration (SLS) (when serum TNF levelspeak), we observed a significant induc-tion of TNF (P � .0001), IL-6 (P �0292), MCP-1 (P � .0007), and

RO-KC (P � .0001) levels in the ma-

FIGURE 2MgSO4 therapy inhibits cytokine anconcentrations in maternal serum

A, TNF, B, IL-6, C, MCP-1, and D, GRO-KC concefter saline or LPS injection (in the presence o

individual data points (●) with geometric meansGRO-KC, growth-related oncogene-KC; IL-6, interleukin-6; LPS, lipopesium sulfate; MLM, MgSO4-LPS-MgSO4; MLS, MgSO4-LPS-salinaline. � and �� indicate P � .05 and P � .01, respectively, com

am Tam. Antenatal MgSO4 ameliorates inflammation. Am

ernal serum when compared with con-

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rol animals (SSS) (Figure 1, A-D).reatment of pregnant animals withgSO4 pre- and post-LPS (MLM) sig-

nificantly decreased LPS-induced TNF(P � .0002), IL-6 (P � .0289), MCP-1(P � .0234), and GRO-KC (P � .0026)levels in the maternal blood at 1½ hrspost-LPS compared with saline-LPStreated animals (SLS) (Figure 1, A-D). Nosignificant reductions in serum inflamma-tory concentrations were observed whenMgSO4 was given only before (MLS) or

nly after LPS treatment (SLM).As expected, the levels of inflamma-

ory mediators (TNF, P � .0002; IL-6,� .0001; MCP-1, P � .0019; and GRO-C, P � .0001) remained significantly

levated in the maternal serum when as-essed 4 hours post-LPS (SLS) comparedith controls (SSS) (Figure 2, A-D).onsistent with the results at 90 minutesost-LPS, we found that maternal treat-ent with MgSO4 (pre- and post-LPS,

chemokineours post-LPS

tions were measured in maternal serum 4 hourssence of MgSO4 therapy). Data are shown as.ccharide; MCP-1, monocyte chemoattractant protein; MgSO4, mag-M, saline-LPS-MgSO4; SLS, saline-LPS-saline; SSS, saline-saline-g SSS to SLS or SLS to either MLM or MLS; # indicates P � .098.

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.0002), IL-6 (P � .0013), and MCP-1(P � .0044) levels in the maternal serum

hen compared with saline-LPS treatednimals (SLS) (Figure 2, A-C). Treat-ent with MgSO4 pre- and post-LPS

MLM) reduced GRO-KC levels in theaternal blood compared with saline-

PS treated dams (SLS) at 4 hours post-PS; however, this was not significantFigure 2, D). MgSO4 treatment wheniven only before LPS administrationMLS) significantly decreased TNF inaternal serum at 4 hours post-LPS by

.1 fold (P � .0296) compared with sa-ine-LPS-treated animals (SLS) (Figure, A). Maternal serum Mg concentra-ions (mg/dL) at 4 hours after saline orPS treatment were as follows: SSS: 2.6 �.52; SLS: 3.0 � 0.84; MLM: 6.067 � 1.89P � .05 vs SLS); MLS: 4.03 � 1.32; SLM:0.5 � 2.85 (P � .001 vs SLS). (MgSO4

was first given as a bolus, followed by se-rial injections once every 20 minutes, asdescribed in the Materials and Methods

FIGURE 3Antenatal MgSO4 administration atconcentrations found in the amnio

A, TNF, B, IL-6, C, MCP-1, and D, GRO-KC concefter saline or LPS injection (in the presence o

individual data points (●) with geometric meansGRO-KC, growth-related oncogene-KC; IL-6, interleukin-6; LPS, lip



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Consistent with previous studies dem-onstrating that maternal infections trig-ger fetal inflammatory responses,8,9 we

bserved a significant induction of TNFP � .0098), IL-6 (P � .0001), MCP-1

(P � .0032), and GRO-KC (P � .0001)in the amniotic fluid 4 hours after mater-nal LPS administration (Figure 3, A-D).Amniotic fluid levels of TNF, IL-6, andMCP-1 (Figure 3) were approximately 8-to 10-fold lower than those observed inmaternal blood (P � .01, P � .01, P �.001 for TNF, IL-6, and MCP-1, respec-tively, Figure 2), whereas amnioticGRO-KC levels were similar to maternalblood concentrations. TNF (P � .0076),IL-6 (P � .0047), MCP-1 (P � .0036),and GRO-KC (P � .006) levels in theamniotic fluid significantly declined inresponse to antenatal MgSO4 therapy

hen administered pre- and post-LPSMLM) (Figure 3, A, B, D) and only am-iotic GRO-KC levels were significantly

ower when MgSO4 was given before LPS

uates proinflammatory mediatorfluid 4 hours post-LPS

tions were measured in amniotic fluid at 4 hourssence of MgSO4 therapy). Data are shown as.ysaccharide; MCP-1, monocyte chemoattractant protein; MgSO4,e; SLM, saline-LPS-MgSO4; SLS, saline-LPS-saline; SSS, saline-, comparing SSS to SLS or SLS to either MLM or MLS.

bstet Gynecol 2011.

njection only (MLS) (P � .0420) com- w

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ared with the saline-LPS-treated ani-als (SLS) (Figure 3, D). Amniotic fluidg concentrations (mg/dL) at 4 hours

fter saline or LPS treatment were as fol-ows: SSS: 2.86 � 0.23; SLS: 3.13 � 0.18;

LM: 3.87 � 0.28 (P � .002 vs SLS);LS: 3.48 � 0.2414; SLM: 3.73 � 0.16.Similar to our observations with cyto-

ines in the amniotic fluid, we found thatPS administration (SLS) significantly

ncreased fetal serum TNF, IL-6, MCP-1,nd GRO-KC levels (assessed 4 hoursost-LPS) when compared with saline-reated control animals (SSS) (Figure 4,-D). At 4 hours post-LPS maternal ad-inistration, TNF, IL-6, and MCP-1 lev-

ls were significantly lower in the fetallood (Figure 4) compared with (1) ma-ernal blood levels (P � .01, P � .01, P �001 for TNF, IL-6, and MCP-1, respec-ively, Figure 2) and (2) amniotic fluidevels of IL-6 (P � .001) and MCP-1 (P �02) (Figure 3). Although fetal serumoncentrations of IL-6 and GRO-KCere reduced in response to MgSO4

treatment (MLM), only fetal serum TNF(P � .0281) levels were significantly de-creased by MgSO4 when administered pre-nd post-LPS (MLM) when comparedith saline-LPS treated animals (SLS)

Figure4,A).Fetalbloodvolumeswere tooow to assess Mg concentrations.

Proinflammatory cytokine expressions elevated within the fetal brain after

aternal LPS administration.27,28 In ourtudy, only MCP-1 and GRO-KC levelsere significantly induced within the

etal brain 4 hours post-LPS (Figure 5,, B). MgSO4, when given pre- and

post-LPS (MLM) significantly reducedGRO-KC levels compared with saline-treated control animals (Figure 5, B).Maternal MgSO4 treatment pre-/post-

PS injection (MLM) reduced fetal brainCP-1 levels (5.4-fold); however, this

eduction was not statistically significantP � .056). By contrast, MgSO4 therapyignificantly attenuated fetal brain

CP-1 concentrations when adminis-ered only before LPS (MLS) (Figure, A).Next, we investigated whether the in-

ibitory effect of MgSO4 on chemokinerotein concentrations was due to re-uced chemokine mRNA expression

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pression within the fetal brain was signif-icantly increased 4 hours after maternalLPS (Figure 5, C) and MgSO4, whengiven pre- and post-LPS (MLM), signif-icantly downregulated LPS-induced fetalbrain MCP-1 mRNA expression (by ap-proximately 3-fold, P � .05) (Figure 5,C) 4 hours post-LPS. Although themRNA expression of GRO-KC and itsreceptor, CXCR2, were not upregulatedwithin the fetal brain after maternal LPSadministration (data not shown), fetalbrain GRO-KC mRNA expression wassignificantly induced 90 minutes post-LPS injection (Figure 5, D). However,MgSO4 treatment (MLM) only slightlyreduced LPS-induced GRO-KC mRNAexpression (P � .187) within the fetalbrain.

To better understand whether MgSO4

directly regulated chemokine produc-tion in the brain, we used a human astro-cyte cell line (U87-MG) to examine theeffects of MgSO4 on MCP-1 and IL-8 (a

XC chemokine family member homol-gous to rat GRO-KC). TNF or IL-1�

stimulation significantly induced bothMCP-1 and IL-8 production (Figure 6,A-D). Treatment of U87-MG cells withMgSO4 significantly reduced TNF-in-duced MCP-1 levels in a dose-dependentmanner (Figure 6, A), but not TNF-in-duced IL-8 levels (Figure 6, B). Treat-ment with MgSO4 significantly reducedL-1�–induced MCP-1 and IL-8 pro-

duction by the neuronal cell line (Figure6, C, D).

COMMENTDespite major research efforts, the inci-dence of preterm births has increased byapproximately 20% over the past 20years and constitutes 12.8% of all livebirths in the United States.29 Severalactors contribute to preterm births, in-luding social stress, genetics, and in-ections, as well as proinflammatory me-iators (eg, TNF, IL-6, IL-8) producedithin the maternal and fetal compart-ents.1,5,30 Survivors of preterm birth

re at increased risk of severe disabilitiesncluding bronchopulmonary dysplasia,etinopathy, neurocognitive impairment,nd cerebral palsy.5,18,30 Using a rat endo-
oxin model of maternal infection-associ-
ted preterm labor, we observed the induc-ion of proinflammatory cytokines (TNFnd IL-6) and chemokines (MCP-1 andRO-KC) in both maternal and fetal com-artments after maternal LPS exposure.e report that maternal administration ofgSO4 significantly reduces proinflam-

matory mediator levels in both maternaland fetal compartments, including the fe-tal brain.

Animal models of endotoxin-inducedpreterm labor mimic the unregulatedmaternal and fetal inflammatory re-sponses observed during maternal infec-tion in humans. Maternal serum andamniotic fluid TNF levels,1 as well as

NF expression in the amnion, chorion,nd decidua31,32 are upregulated inomen presenting in labor. Consistentith these observations, the injection ofNF induces preterm labor in experi-ental animals.33 Likewise, elevated ma-

ternal serum, fetal plasma, and amniotic

FIGURE 4Maternal MgSO4 reduces cytokinein fetal blood 4 hours after matern

A, TNF, B, IL-6, C, MCP-1, and D, GRO-KC concealine or LPS injection (in the presence of absen

data points (●) with geometric means (�).GRO-KC, growth-related oncogene-KC; IL-6, interleukin-6; LPS, lip



fluid IL-6 levels are linked to the onset of

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labor.1,34-36 MCP-1, a chemoattractantrotein for monocytes and other leuko-ytes, is proposed to play an importantole in preterm labor by promoting thenflux of monocytes into the myome-rium, cervix, and fetal membranes justefore the initiation of parturition,37 and

ncreased MCP-1 concentrations in ges-ational tissues and amniotic fluid areinked to preterm labor in humans.38-40

CXCL1 (GRO-�), known as GRO-KC inodents, is a neutrophil chemoattractantnduced during endotoxemia.41 CXCL1ithin the amniotic fluid is a potential aarker of intraamniotic infections in

umans.42

Consistent with previous rodent stud-ies,8,9,43,44 we found that maternal LPSadministration significantly induced cy-tokine production in maternal and fetalcompartments. More specifically, wefound that LPS increased TNF, IL-6,MCP-1, and GRO-KC levels in maternal

chemokine concentrationsPS

tions were measured in fetal blood 4 hours afterf MgSO4 therapy). Data are shown as individual

ysaccharide; MCP-1, monocyte chemoattractant protein; MgSO4,e; SLM, saline-LPS-MgSO4; SLS, saline-LPS-saline; SSS, saline-, comparing SSS to SLS or SLS to MLM.

bstet Gynecol 2011.

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(comprised mainly of fetal urine andlung secretions45)(Figure 3), and fetalblood (Figure 4), with the greatest in-creases observed in the maternal blood(probably reflecting the systemic mater-nal inflammation induced after ip LPSadministration). At 4 hours post-LPS,amniotic fluid levels of IL-6 (P � .001)nd MCP-1 (P � .02) (Figure 3) wereignificantly higher than IL-6 and

CP-1 levels found in fetal blood at 4ours (Figure 4). Of the 4 mediators ex-mined after maternal LPS injection,nly MCP-1 and GRO-KC were detect-ble in the fetal brain (Figure 5). Inter-stingly, both neural MCP-1 andRO-KC expression are associated witheutrophil-mediated brain damage dur-

ng inflammation46 and increased per-eability of the blood brain barrier.47-49

FIGURE 5MgSO4 therapy alters chemokine cin the fetal brain 4 hours after mat

A and B, Fetal brain chemokine concentrations (homogenates at 4 hours after saline or LPS injecpoints (●) with geometric means (�), corrected f05 comparing SSS to SLS, or SLS to either MLeasured by Q-PCR at 4 hours after saline oeasured by Q-PCR at 90 minutes after saline

comparing SSS to SLS or SLS to MLM; # indicatGRO-KC, growth-related oncogene-KC; IL-6, interleukin-6; LPS, lip

agnesium sulfate; MLM, MgSO4-LPS-MgSO4; MLS, MgSO4-LPSaline-LPS-MgSO4; SLS, saline-LPS-saline; SSS, saline-saline-sa


Our inability to detect LPS-induced TNF t


or IL-6 proteins in fetal brains 4 hoursafter maternal LPS injection might haveresulted from using brain homogenatesrather than immunostaining of neuronaltissue sections and/or using 1 time pointfor assessment (4 hours post-LPS).However, our results are consistent withprevious reports that detected only TNFmRNA transcripts (not protein) in fetalbrains.50,51 Finally, our study did not as-ess preterm birth nor did we measureinjury” in the fetal brains. Therefore, were unable to conclude that maternalgSO4 administration protects against

PS-induced preterm birth or brain in-ury in rat offspring.

In addition to elucidating the patho-enesis underlying infection- and in-ammation-mediated preterm labornd poor neonatal outcome, experimen-

centrations and expressional LPS administration

-1, A, and GRO-KC, B) were measured in brain(� MgSO4). Data are shown as individual data

rotein concentrations. A and B, � indicates P �r MLS. C, Fetal brain MCP-1 mRNA expressionPS. D, Fetal brain GRO-KC mRNA expressionPS (� MgSO4). C and D, � indicates P � .05� .056.

ysaccharide; MCP-1, monocyte chemoattractant protein; MgSO4,e; Q-PCR, quantitative real-time polymerase chain reaction; SLM,

bstet Gynecol 2011.

al models can be used to test interven-

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ions (eg, antibiotics, progesterone, andL-10).11-13 Antenatal MgSO4 adminis-ration targeted multiple cytokines andhemokines in both maternal and fetalompartments associated with pretermabor in this model and was most effec-ive when given pre- and post-LPS prob-bly because it accumulated in eachompartment prior to “infection,” withhe greatest effect in the maternal com-artment. Given the strong associationetween the elaboration of proinflam-atory mediators and the activation of

he prostaglandin cascade within the ges-ational tissues as a mechanism for pre-erm parturition, we hypothesize thathe tocolytic effects of MgSO4 could bettributed, in part, to its suppression ofhis response. Our observations showinghat antenatal MgSO4 treatment reduced

endotoxin-mediated inflammation inmaternal and fetal compartments areconsistent with studies by Hallak et al25

describing the protective effects ofMgSO4 in models of hypoxia-inducedfetal brain damage, and more recently,Burd et al51 revealing the neuroprotec-tive effects of MgSO4 in LPS-mediatedetal brain damage. Several notable dif-erences distinguish our study from thatf Burd et al51: (1) we examined the ef-

fects of maternal MgSO4 administrationn multiple inflammatory mediatorscytokines and chemokines) within bothaternal serum, amniotic fluid, fetal

lood, and fetal brain rather than fetalrains alone; (2) we used a systemic ma-ernal infection model rather than a lo-alized (intrauterine) infection model;3) we measured inflammatory proteinevels rather than mRNA transcripts; and4) we used rats instead of mice. To-ether, these data support the overallrotective and antiinflammatory effects ofaternal MgSO4 administration on fetal

and maternal responses and we hypothe-size that MgSO4 therapy may exert neuro-protective effects in rat offspring similar tothat observed in humans.19-21,52

Our findings are potentially importantgiven the link between Mg deficiency andinflammation. Mg deficiency is associ-ated with numerous conditions, includ-ing bacterial infections,53,54 generalizednflammation,55-57 and increased blood

onern

MCPtion

or pM or Lor Les Popol

-salinline.

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brain barrier permeability during sepsis.58

cam


Despite recommendations for pregnantwomentoconsumeadditionalMgtocom-pensate for maternal losses because of thedemands of growing maternal and fetal tis-sues,59 several studies indicate insufficientMg intake during pregnancy.60-62 Mg defi-iency during pregnancy has been associ-ted with early parturition and compro-ised fetal growth/development.63 Thus,

we hypothesize that maternal Mg admin-istration during maternal infection couldsuppress damaging inflammatory media-tor production within the maternal and fe-tal compartments, including the fetalbrain, to improve birth outcome. Futurestudies will be designed to test ourhypotheses. f

ACKNOWLEDGMENTSWe thank Meredith Akerman of the BiostatisticsCore of the Feinstein Institute who performedthe statistical analyses, the staff of the Centerfor Comparative Physiology of the Feinstein In-

FIGURE 6MgSO4 significantly suppresses TNMCP-1 production by U87-MG cells

U87-MG cells (human astrocyte cell line) were treatB, vehicle treatment or TNF (50 ng/mL) stimulation oovernight incubation, A, C, MCP-1 and B, D, IL-8Data are shown as mean chemokine concentrations.001 comparing vehicle to TNF- or IL-1��treatedMgSO4 vs TNF- or IL-1��treated cells in the presMCP-1, monocyte chemoattractant protein; MgSO4, magnesium


stitute who assisted with the animal experimen-

tation, and Dr Ravi Shankar, MD and Ms SarahHan for critically reading the manuscript.

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Magnesium sulfate ameliorates maternal and fetal inflammation in a rat model of maternal infection