Research Article Peripheral Inhibitor of AChE, Neostigmine, Prevents the Inflammatory Dependent ...

13 Pages·2017·1.53 MB·English

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BioMed Research International
Volume 2017, Article ID 6823209, 12 pages
https://doi.org/10.1155/2017/6823209
Research Article
Peripheral Inhibitor of AChE, Neostigmine, Prevents the
Inflammatory Dependent Suppression of GnRH/LH Secretion
during the Follicular Phase of the Estrous Cycle
AndrzejP.Herman,1JaninaSkipor,2AgataKrawczyNska,1JoannaBochenek,1
KarolinaWojtulewicz,1HannaAntushevich,1AnnaHerman,3KamilaPaczesna,1
KatarzynaRomanowicz,1andDorotaTomaszewska-Zaremba1
1TheKielanowskiInstituteofAnimalPhysiologyandNutrition,PolishAcademyofSciences,Jabłonna,Poland
2InstituteofAnimalReproductionandFoodResearch,PolishAcademyofSciences,Olsztyn,Poland
3FacultyofCosmetology,TheAcademyofCosmeticsandHealthCare,Warsaw,Poland
CorrespondenceshouldbeaddressedtoAndrzejP.Herman;[email protected]
Received 3 May 2017; Revised 6 July 2017; Accepted 16 July 2017; Published 15 August 2017
AcademicEditor:DanieleTomassoni
Copyright©2017AndrzejP.Hermanetal. This is an open access article distributed under the Creative Commons Attribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
The study was designed to test the hypothesis that the inhibition of acetylcholinesterase (AChE) activity at the periphery by
Neostigmine (0.5mg/animal) will be sufficient to prevent inflammatory dependent suppression of the gonadotropin-releasing
hormone(GnRH)/luteinisinghormone(LH)secretioninewesinthefollicularphaseoftheestrouscycle,andthiseffectwillbe
comparablewiththesystemicAChEinhibitor,Donepezil(2.5mg/animal).Animmune/inflammatorychallengewasinducedby
peripheraladministrationoflipopolysaccharide(LPS;400ng/kg).PeripheraltreatmentwithDonepezilandNeostigmineprevented
theLPS-induceddecrease(𝑃 < 0.05)inLH𝛽geneexpressionintheanteriorpituitarygland(AP)andinLHrelease.Moreover,
Donepezilcompletelyabolished(𝑃<0.05)thesuppressoryeffectofinflammationonGnRHsynthesisinthepreopticarea,when
pretreatment with Neostigmine reduced (𝑃 < 0.05) the decrease in GnRH content in this hypothalamic structure. Moreover,
administrationofbothAChEinhibitorsdiminished(𝑃<0.05)theinhibitoryeffectofLPStreatmentontheexpressionofGnRH
receptorintheAP.OurstudyshowsthatinflammatorydependentchangesintheGnRH/LHsecretionmaybeeliminatedorreduced
byAChEinhibitorssuppressinginflammatoryreactiononlyattheperipherysuchasNeostigmine,withouttheneedforinterfering
inthecentralnervoussystem.
1.Introduction formaintainingthehomeostasis.Thehypothalamusplaysa
key role in the control of reproduction in females by tonic
Animmune/inflammatorychallengescausedbythebacterial release of gonadotropin-releasing hormone (GnRH) to the
orviralinfectioncouldbeoneofthereasonsofreproductive hypothalamic-pituitary portal circulation. In turn, GnRH
disordersinbothhumansandanimals[1].Itispostulatedthat regulates the secretion of luteinising hormone (LH) and
the interaction between the immune and neuroendocrine follicle-stimulating hormone (FSH) from the gonadotropic
systemsmayoccuratalllevelsoftheneurohormonalsystem cellsintheanteriorpituitarygland(AP)[2].
of hypothalamic-pituitary-gonadal (HPG) axis controlling Itwaspreviouslyreportedthatbothacuteandprolonged
the female reproductive process. A particularly important inflammation induced by peripheral administration of bac-
role in the communication between these two systems is terial endotoxin-lipopolysaccharide (LPS) may disturb the
played by the hypothalamus, the part of the brain respon- secretionofGnRHandLH[3,4].Thestudyonewesinthe
sible for the integration and processing of signals from the follicularphaseoftheestrouscycleshowedthatinflammation
nervous, endocrine, and immune systems, what is essential interrupted the preovulatory estradiol increase and delayed
2 BioMedResearchInternational
orblocks thesubsequent LHand FSHsurges [5]. Thissup- willbesufficienttopreventtheLPS-inducedsuppressionof
pressiveeffectofinflammationonthegonadotropinssecre- GnRH/LH secretion in ewes in the follicular phase of the
tion seems to be mediated via proinflammatory cytokines estrous cycle, and this effect will be comparable with the
reaching the hypothalamic area during immune challenges systemicactionofDonepezil.
[6].Interleukin-(IL-)1𝛽andtumornecrosisfactor𝛼(TNF𝛼)
mayrepresentthemajorproinflammatorycytokinesmediat-
2.MaterialsandMethods
ing the LPS-induced suppression of GnRH and LH release,
whereastheroleofIL-6inthisprocessseemstobemarginal
2.1. Animals. The studies were performed on adult, 2-
[6–8].
year-old Blackhead ewes during the reproductive season
One of the endogenous mechanisms involved in the
(September-October). The ewes were maintained in good
regulation of immune response and cytokine secretion is
conditions; that is, their body condition was estimated at 3
the cholinergic anti-inflammatory pathway. It had been
inafive-pointscale[14]andtheanimalswereacclimatedto
previouslydescribedthatthecholinergicanti-inflammatory
the experimental conditions for one month. The ewes had
pathwaycouldbeactivatedbystimulationofthevagusnerve
constant visual contact with each other in order to avoid
thereby increasing the acetylcholine (ACh) secretion [9].
isolationstress.Theanimalswerefedaconstantdietofcom-
This anti-inflammatory mechanism could be also activated
mercialconcentrateswithhayandwateravailableadlibitum,
by pharmacological blockade of the acetylcholinesterase
accordingtotherecommendationsproposedbytheNational
(AChE)activity,theenzymeresponsibleforthedegradation
ResearchInstituteofAnimalProductionforadultewes[15].
ofACh.InvitrostudiesrevealedthatAChactingprobablyvia
Inordertobeststandardizeexperimentalconditionsthe
nicotinicreceptorCHRNA7reducedLPS-stimulatedrelease
of proinflammatory cytokines, including IL-1𝛽, IL-6, and stage of the estrous cycle of ewes were synchronized by the
TNF𝛼 [10]. In vivo study also showed that blockade of Chronogest(cid:2) CR (Merck Animal Health, Boxmeer, Nether-
AChE activity reduced synthesis of IL-1𝛽 during peripheral lands) method using an intravaginal sponge impregnated
with20mgofasyntheticprogesterone-likehormone.Allewe
inflammation in mouse [11] and sheep [12] hypothalamus.
hadChronogestCRspongesplacementfor14days.Following
Moreover, our previous study on ewes showed that the
spongeremoval,theeweswillreceiveanintramuscularinjec-
activation of the cholinergic anti-inflammatory pathway
tionof500iupregnantmare’sserumgonadotropin(PMSG)
by Rivastigmine may abolish the inhibitory effect of LPS
(Merck Animal Health, Boxmeer, the Netherlands). The
administrationontheGnRH/LHsecretionandreducedthe
experimentalprocedurewasperformed24hfollowingPMSG
releaseofstressmarkerssuchascortisolandprolactin[13].
injection.Intreatedanimals,theimmunestresswasinduced
However, Rivastigmine, AChE inhibitor used in this study,
by the intravenous (iv.) injection of LPS from Escherichia
exhibits the systemic action; therefore, it blocks the AChE
coli 055:B5 (Sigma-Aldrich, St. Louis, MO, USA) in a dose
activitybothinthebrainparenchymaandintheperiphery,
of 400ng/kg, dissolved in saline (0.9%w/v NaCl) (Baxter,
because it easily crosses the blood-brain barrier (BBB).
Deerfield,IL,USA)ataconcentrationof10mg/L.
Therefore, it could not be concluded whether and to what
extenttheobservedreductionofIL-1𝛽synthesisinthecentral All procedures were performed with agreement of the
Local Ethics Committee of Warsaw University of Life
nervous system (CNS) and changes in hormone secretion
Sciences-SGGW.
resulted from the inhibition of the AChE activity in the
CNSorthereductioninperipherallevelsofproinflammatory
cytokines. The results of experiments performed on mice 2.2. Experimental Procedures. Venous catheters were im-
suggestthatonlythereductionofcirculatingconcentrationof planted into thejugular vein on the day priorto theexper-
proinflammatorycytokinesundercertainconditionsmaybe iment.Ewes(𝑛 = 36)wererandomlydividedintosixexper-
sufficient to significant inhibition of LPS-induced synthesis imentalgroups(Table1).Jugularbloodsamples(6ml)were
ofIL-1𝛽intheCNS[11].Thisstudysuggeststhat,todisturb takenformeasurementoftheperipheralhormoneat15min
thefunctioningofCNS,thebloodlevelofimmunemediators intervals beginning 2h before the iv. administration of LPS
hastoenrichacriticallevel.Therefore,thereductionofproin- or an equivalent volume of saline injection and continuing
flammatorycytokineconcentrationbelowthiscriticalvalue for3h.HalfhourpriortoLPS/salinetreatmenttheanimals
mayblockthetransmissionoftheinflammatorysignalinto were slowly intravenously treated with saline (groups 1 and
the brain parenchyma. These all suggest that the activation 2)orsuitableAChEinhibitor(groups3,4,5,and6)(Table1).
of the cholinergic anti-inflammatory pathway only in the After the blood collection, the animals were immediately
peripherymaybesufficienttostopexcessiveincreaseinthe euthanized (3h after LPS or saline administration) and the
concentration of proinflammatory cytokines in the blood, brains were rapidly removed from the skulls. From the
whichinturnmaybesufficienttoreversethenegativeeffects ovine brains four hypothalamic structures were dissected
of immune stress on the GnRH/LH, without providing the due to theirinvolvementin theGnRH-ergic activity.In the
AChEinhibitoranddirectinterferenceintheCNS.Therefore, hypothalamus of sheep GnRH-ergic neurons did not form
inthepresentstudyweusedtwoAChEinhibitorsdifferingin dense clusters, but they spread from brain septum and the
theabilitytocrosstheBBB:Donepezilwhichgreatlycrossthe horizontal diagonal band of Broca, through the preoptic
BBBandNeostigminewhichdoesnotpenetratetheBBB. area(POA),anteriorhypothalamus(AHA),andmedialbasal
The present study tested the hypothesis that the hypothalamus(MBH)[2].However,mostofGnRHneurons
inhibitionofAChEactivityattheperipherybyNeostigmine havetheirpericarionslocatedinthePOA;therefore,itplaysa
BioMedResearchInternational 3
Table1:Theschemeoftheexperiment.
Experimental Experimental
Dose Dose
Group Numberofanimals treatmentI [mg/animal] treatmentII [ng/kg]
(iv.) (iv.)
1:control 6 NaCl 0 NaCl 0
2:LPStreated 6 NaCl 0 LPS 400
3:Donepeziltreated 6 Donepezil 2.5 NaCl 0
4:Neostigminetreated 6 Neostigmine 0.5 NaCl 0
5:Donepezil+LPStreated 6 Donepezil 2.5 LPS 400
6:Neostigmine+LPStreated 6 Neostigmine 0.5 LPS 400
Totalamountofanimals 36
pivotalroleinGnRHsynthesis.ThemajorityofGnRH-ergic accordingtoKokotandStupnicki[23],usingrabbitanticor-
neuronssendtheiraxonalprojectiontothemedianeminence tisol antisera (R/75) and an HPLC-grade cortisol standard
(ME) where GnRH is released to the hypophyseal portal (Sigma-Aldrich, St. Louis, MO, USA). The assay sensitivity
system [2]. The hypothalamic structures such as the POA, was 1ng/ml and the intra- and interassay coefficients of
AHA,MBH,andMEweredissectedaccordingtostereotaxic variationforcortisolwere9%and12%,respectively.
atlasofthesheepbrain[18]asitwasdescribedelsewhere[13].
Landmarks were the mammillary body, median eminence, 2.3.5.ELISAAssayfortheGnRHConcentrationinthePOA.
and optic chiasm. The depths of the cuts were 2 to 2.5mm The concentrations of GnRH in the POA were determined
for MBH and 2.5 to 3mm for AHA and POA. All tissues withacommercialGnRHELISAkit(BlueGeneBiotechCo.,
were frozen immediately after collection in liquid nitrogen Ltd.,China)dedicatedforsheep.AllstagesofGnRHanalysis
andthenwillbestoredat−80∘C. wereperformedaccordingmanufacturer’sprotocol.Thetis-
sueswerehomogenizedin400𝜇lofphosphatebufferedsaline
2.3.Assays (0.02M). Then homogenates were subjected to two freeze-
thawcyclestofurtherbreakthecellmembranes.Afterthat,
2.3.1. Radioimmunoassay for LH. The plasma LH concen- thehomogenateswerecentrifugatedfor15minat1500×gin
tration was assayed with a double-antibody RIA using ∘
4 C.Thesupernatantswerealiquotedandstoreduntilassay
anti-ovine-LHandanti-rabbit-𝛾-globulinantiseraandovine in−80∘C.Allstepsintheassayswereperformedaccordingto
standard (teri.oLH, Tucker Endocrine Research Institute),
themanufacturer’sinstructions.Theincubationofplatesand
accordingtoStupnickiandMadej[19].Theassaysensitivity
absorbancemeasurementat450nmwereperformedusinga
was 0.3ng/ml and the intra- and interassay coefficients of
VersaMaxreader(MolecularDevicesLLC,Sunnyvale,Cali-
variationwere8%and11.5%,respectively.
fornia,UnitedStates).Theassaysensitivitywas1.0pg/ml.The
valuesofGnRHconcentrationwerenormalisedtototalpro-
2.3.2. Radioimmunoassay for FSH. The concentration of teincontentineachsampleassayedusingBradfordmethod.
FSHwasdeterminedbydouble-antibodyradioimmunoassay
(RIA)usinganti-ovine-FSH(teri.anti-oFSH)andanti-rabbit-
2.3.6.DeterminingtheRelativeGeneExpression. TotalRNA
𝛾-globulin antisera, according to L’Hermite et al. [20]. The
from the hypothalamic structure and AP were isolated
anti-FSH, as well as the FSH standard (teri. oFSH-and teri. using the components of NucleoSpin(cid:2) RNA/Protein Kit
FSH ig), was kindly supplied by Dr. Reichert Jr. (Tucker
(MACHEREY-NAGEL Gmbh & Co., Du¨ren, Germany)
Endocrine Research Institute LLC, Atlanta, Georgia, USA).
according to a manufacturer’s instruction. The purity and
Theassaysensitivitywas1.5ng/mlandtheintra-andinteras-
concentrationofisolatedRNAwerespectrophotometrically
saycoefficientsofvariationwere3.5%and11.3%,respectively.
quantifiedbymeasuringtheopticaldensityat230,260,and
280nm in a NanoDrop 1000 instrument (Thermo Fisher
2.3.3.RadioimmunoassayforProlactin. Theplasmaprolactin Scientific Inc., Waltham, USA). The RNA integrity was
concentration was assayed by a radioimmunoassay double- verifiedbyelectrophoresisusing1%agarosegelstainedwith
antibodymethod,usingspecificantiovineprolactinandanti- ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA).
rabbit-𝛾-globulin antisera according to Wolin´ska et al. [21]. Maxima(cid:3) First Strand cDNA Synthesis Kit for RT-qPCR
Theprolactinstandardforiodinationwasobtainedaccording (ThermoFisherScientificInc.,Waltham,USA)wasusedto
to the method described by H. Kochman and K. Kochman preparecDNAsynthesis.AsastartingmaterialforthisPCR
[22].Theassaysensitivityforprolactinwas2ng/ml,andthe synthesis2𝜇goftotalRNAwasused.
intra- and interassay coefficients of variation were 9% and Real-timeRT-PCRwascarriedoutusingHOTFIREPol
12%,respectively. EvaGreen(cid:2) qPCR Mix Plus (Solis BioDyne, Tartu, Estonia)
components and HPLC-grade oligonucleotide primers syn-
2.3.4. Radioimmunoassay for Cortisol. The cortisol con- thesised by Genomed (Poland), according to the method
centrations were determined by radioimmunoassay (RIA) described elsewhere [16]. Specific primers for determining
4 BioMedResearchInternational
the expression of housekeeping genes and the genes of number sc-47778, Santa Cruz Biotechnology Inc., Dallas,
interestwerechosenbasedonourpreviousstudies(Table2). USA)dissolvedinblockingbufferatdilutionsof1:500and
Onetubecontained4𝜇lPCRMasterMix(5x),14𝜇lRNase- 1:1000,respectively.Afterwashingthreetimes,membranes
free water, 1𝜇l primers (0.5𝜇l each, working concentration were incubated with the following secondary HRP conju-
was 0.25𝜇M), and 1𝜇l cDNA template. The tubes were run gated antibodies: donkey anti-goat IgG-HRP (cat. number
on the Rotor Gene 6000 (Qiagen, Duesseldorf, Germany). sc-2304, Santa Cruz Biotechnology Inc., Dallas, TX, USA)
∘
Thefollowingprotocolwasused:95 Cin15minforactivating and goat anti-mouse IgG1 heavy chain (HRP) (cat. number
HotStarDNApolymeraseandfinallythePCRincluding30 ab97240, Abcam, Cambridge, UK) dissolved in blocking
∘ ∘
cycles at 95 C in 10sec for denaturation, 60 C in 20sec for buffer at a dilution of 1:10,000. After washing three times,
∘
annealing,and72 Cin10secforextension.Afterthecycles, themembraneswerevisualisedusingchromogenicdetection
afinalmeltingcurveanalysisundercontinuousfluorescence withaPierce1-stepTMB-blottingsubstratesolution(Thermo
measurementswasperformedtoconfirmthespecificityofthe Fisher Scientific, Waltham, MA, USA). After visualisation,
amplification. themembranesweredriedandscannedusinganEpsonPer-
Relative gene expression was calculated using the com- fectionV370Photoscanner(SeikoEpsonCorporation,Suwa,
parative quantification option [24] of the Rotor Gene Japan). Densitometric analysis of the scanned membrane
6000 software version 1.7 (Qiagen, Dusseldorf, Germany). wasperformedusingthesoftwareImageJ(ResearchServices
Threehousekeepinggeneswereexamined:glyceraldehyde-3- Branch,NationalInstituteofMentalHealth,Bethesda,MD,
phosphate dehydrogenase (GAPDH), 𝛽-actin (ACTB), and USA).
histone deacetylase 1 (HDAC1). The mean expression of
these three housekeeping genes was used to normalise the
2.4.StatisticalAnalysisofData. Theresultsofhormonescon-
expression of the analysed genes. The results are presented
centrationarepresentedasthemean±SEM.Allexperiments
inarbitraryunits,astheratioofthetargetgeneexpressionto
consistedofabaselineperiodwhennotreatmentwasgiven
themeanexpressionofthehousekeepinggenes.
(2 to 0.5h before) and a period after treatment (1 to 3h
after).Toidentifytreatmenteffects,themeanvaluesforthe
2.3.7. Western Blot Assays for GnRHR Expression in the
baseline and treatment periods were obtained. To compare
AP. Before electrophoresis, the protein concentrations of
thebaselineperiodwhennotreatmentwasgivenandaperiod
samples isolated previously from the AP using the Nucle-
aftertreatment,theobtaineddatawerecomparedwithuseof
oSpinRNA/ProteinKit(MACHEREY-NAGELGmbh&Co., Student’s𝑡-testfordependentsamples(“repeatedmeasures”).
Du¨ren,Germany)werequantifiedusingaProteinQuantifi- Statisticalsignificancewasdefinedas𝑃<0.05.
cationAssayKit(MACHEREY-NAGELGmbh&Co.,Du¨ren,
The results of blood hormones concentration obtained
Germany).Theappropriatevolumeofmoleculargradewater
only after treatment period, GnRH content in the POA,
(Sigma-Aldrich,St.Louis,MO,USA)wasaddedtoavolume
GnRHRproteinexpression,andallexaminedgenesexpres-
ofsamplecontaining50𝜇goftotalproteintobringthetotal
sion were analysed using a two-way ANOVA, the exam-
samplevolumeto20𝜇l.Next,19𝜇lofLaemmlibuffer(Sigma-
ined factors were inflammatory state and AChE inhibitor
Aldrich,St.Louis,MO,USA)and1𝜇lof𝛽–mercaptoethanol
treatment(DonepezilorNeostigmine).BeforeANOVAwas
(Sigma-Aldrich, St. Louis, MO, USA) were added. Such
conducted its two assumptions were checked: normality
mixtures were boiled for 3min. Electrophoresis was then
(Shapiro-Wilk’stest)andhomogeneityofthevariances(Lev-
performed in the presence of molecular weight markers
ene’stest).Whenasignificanttreatmentbytimeinteraction
(Spectra Multicolor Broad Range Protein Ladder, Thermo
wasobserved,aposthocanalysiswasconductedtoidentify
FisherScientificInc.,Waltham,MA,USA).Denaturedsam-
treatmenteffects.Fisher’sleastsignificantdifferenceposthoc
plesandmolecularweightstandardswereloadedonto4–12%
test was used to compare precompared with posttreatment
polyacrylamide gels and subjected to electrophoresis in a values.Statisticalsignificancewasdefinedas𝑃<0.05.
Tris-glycinerunningbufferusingtheProteanIIxiCell(Bio-
ThestatisticalanalysiswasperformedusingtheSTATIS-
RadLaboratories,Inc.,Hercules,CA,USA)accordingtothe
TICA10software(StatSoftInc.,Tulsa,OK,USA).
manufacturer’sinstructions.Next,proteinsweretransferred
in Tris-glycine blotting buffer to polyvinylidene difluoride
membranes(Immobilon(cid:3)-P(0.45𝜇m),MerckKGaA,Darm- 3.Results
stadt,Germany)usingtheTrans-Blot(cid:2)SDSemi-DryTransfer
Cell (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 3.1. Effect of AChE Inhibitors and LPS Injection on LH,
30minat20V.Themembraneswereblockedfor1hatroom FSH, Prolatin, and Cortisol Release. Both Donepezil and
temperature in blocking buffer made up of Tris buffered NeostigminetreatmentpreventedtheLPS-induceddecrease
salineatpH7.5with0.05%Tween-20(TBST)(Sigma-Aldrich, (𝑃 < 0.05) in plasma concentrations of LH. Moreover,
St. Louis, MO, USA) containing 3% bovine serum albumin in animals treated with Neostigmine and LPS the plasma
fractionV(Sigma-Aldrich,St.Louis,MO,USA).Next,mem- concentration of LH was higher (𝑃 < 0.05) than in
∘
branes were incubated overnight at 4 C with the following the control group (Figure 1(a)). In contrast, the peripheral
primary antibodies: goat anti-GnRHR polyclonal antibody concentrations of FSH were unaffected by all treatments
(cat.numbersc-8682,SantaCruzBiotechnologyInc.,Dallas, (Figure 1(b)). Endotoxin injection increased (𝑃 < 0.05)
USA) and mouse anti-ACTB monoclonal antibody (cat. the concentration of stress markers: cortisol (Figure 2(a))
BioMedResearchInternational 5
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Table2:Allgenesanalyzedb GeneGAPDHglyceraldehyde-3-phosphatedehydrogenaseACTBbetaactinHDAC1histonedeacetylase1GnRHRgonadotropin-releasinghormonereceptorGnRHgonadotropin-releasinghormoneLHBluteinizinghormonebeta-subunitFSHBfolliclestimulatinghormonebeta-subunitPRLprolactin
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6 BioMedResearchInternational
4 90 ＂∗
＂∗
＃∗ 80 ＂∗
g/ml) 23 B B B ！∗＂＃∗ ng/ml) 567000
LH (n 1 ortisol ( 3400 A
C 20 A
A
0 10
Before After 0
Before After
Control LPS
Donepezil Donepezil + LPS Control LPS
Neostigmine Neostigmine + LPS Donepezil Donepezil + LPS
Neostigmine Neostigmine + LPS
(a)
(a)
12 A
A
250
10 A A A A ＂∗
ml) 8 ml) 200 ＂∗ ＂∗
ng/ 6 ng/ 150
H ( n (
FS 4 cti 100
a
2 Prol 50 A
A A
0
Before After 0
Before After
Control LPS
Donepezil Donepezil + LPS Control LPS
Neostigmine Neostigmine + LPS Donepezil Donepezil + LPS
Neostigmine Neostigmine + LPS
(b)
(b)
Figure 1: Effect of lipopolysaccharide (LPS; 400ng/kg; iv.) and Figure 2: Effect of lipopolysaccharide (LPS; 400ng/kg; iv.) and
acetylcholinesteraseinhibitors:Donepezil(2.5mg/animal;iv.)and acetylcholinesteraseinhibitors:Donepezil(2.5mg/animal;iv.)and
Neostigmine(0.5mg/animal;iv.)injectionsonbloodconcentration Neostigmine(0.5mg/animal;iv.)injectionsonbloodconcentration
ofluteinisinghormone(LH)(a)andfollicle-stimulatinghormone ofstressmarkers:cortisol(a)andprolactin(b)concentrationinthe
(FSH)(b)concentrationinthebloodplasma.Thedataarepresented blood plasma. The data are presented as the mean value ± SEM.
asthemeanvalue±SEM.Allexperimentsconsistedofabaseline Allexperimentsconsistedofabaselineperiodwhennotreatment
period when no treatment was given (2 to 0.5h before) and a was given (2 to 0.5h before) and a period after treatment (1 to
periodaftertreatment(1to3hafter).∗:asteriskindicatesstatistically 3h after). ∗: asterisk indicates statistically significant differences
significantdifferencesbetweentheperiodwhennotreatmentwas between the period when no treatment was given and a period
given and a period after treatment according to Student’s t-test aftertreatmentaccordingtoStudent’s𝑡-testfordependentsamples
fordependentsamples(“repeatedmeasures”).Theresultsofblood (“repeatedmeasures”).Theresultsofbloodhormonesconcentration
hormonesconcentrationobtainedonlyaftertreatmentperiodwere obtained only after treatment period were analysed using a two-
analysedusingatwo-wayANOVA.Differentcapitallettersindicate wayANOVA.Differentcapitallettersindicatesignificantdifferences
significantdifferencesaccordingtoatwo-wayANOVAfollowedby accordingtoatwo-wayANOVAfollowedbyFisher’sposthoctest.
Fisher’sposthoctest.Statisticalsignificancewasdefinedas𝑃<0.05. Statisticalsignificancewasdefinedas𝑃<0.05.
3.3. Effect of AChE Inhibitors and LPS Injection on GnRHR
and prolactin (Figure 2(b)), and these increases were not Protein Expression in the AP. Inflammation reduced (𝑃 <
influencedbytheAChEinhibitorstreatment. 0.05) expression of GnRHR in the AP of ewes but the
precedinginjectionofDonepezilandNeostigmineabolished
3.2. Effect of AChE Inhibitors and LPS Injection on GnRH theinhibitoryeffectofLPStreatmentontheexpressionofthis
Content in the POA. Endotoxin treatment decreased (𝑃 < receptor(Figure4).
0.05)thecontentofGnRHinthePOA.Precedinginjection
of Donepezil completely abolished this suppressory effect 3.4. Effect of AChE Inhibitors and LPS Injection on the
of inflammation on the GnRH content in the POA, when Gene Expression in the Hypothalamus and AP. Endotoxin
pretreatment with Neostigmine reduced (𝑃 < 0.05) the treatment decreased (𝑃 < 0.05) the level of GnRH mRNA
negativeeffectofinflammationontheGnRHcontentinthe only in the ME, but preceding injection of both AChE
POA, but it stayed significantly lower compared with the inhibitors prevented this effect of inflammation. It is worth
controlgroup(Figure3). mentioning that the gene expression of GnRH was not
BioMedResearchInternational 7
20 1.2
18 C C on B B
16 BC BC essi 1 B
mg) 14 B expr B B
nRH (pg/ 116802 A R protein 00..68 A
G H
4 R 0.4
n
2 G
0 ative 0.2
el
Control LPS R 0
Donepezil Donepezil + LPS
Neostigmine Neostigmine + LPS Control LPS
Donepezil Donepezil + LPS
Figure 3: Effect of lipopolysaccharide (LPS; 400ng/kg; iv.) and
Neostigmine Neostigmine + LPS
acetylcholinesteraseinhibitors:Donepezil(2.5mg/animal;iv.)and
Neostigmine(0.5mg/animal;iv.)injectionsonbloodthecontentof GnRHR (36kDa)
gonadotropin-releasinghormone(GnRH)inthehypothalamusof
ewesduringthefollicularphaseoftheestrouscycle.Thedataare
presentedasthemeanvalue±SEM.Theresultswereanalysedusing ACTB (42kDa)
a two-way ANOVA. Different capital letters indicate significant
differencesaccordingtoatwo-wayANOVAfollowedbyFisher’spost Figure 4: Effect of lipopolysaccharide (LPS; 400ng/kg; iv.) and
hoctest.Statisticalsignificancewasdefinedas𝑃<0.05. acetylcholinesteraseinhibitors:Donepezil(2.5mg/animal;iv.)and
Neostigmine (0.5mg/animal; iv.) injections on the relative pro-
tein expression (mean ± SEM; 𝑛 = 6 animals per group) of
gonadotropin-releasinghormonereceptor(GnRHR)intheanterior
affected by any treatment in other hypothalamic structures pituitary of ewes during the follicular phase of the estrous cycle.
analysed(Table3). Thedataarepresentedasthemeanvalue±SEM.Theresultswere
analysedusingatwo-wayANOVA.Differentcapitallettersindicate
In the AP, the inflammationdecreased the gene expres-
significantdifferencesaccordingtoatwo-wayANOVAfollowedby
sionofGnRHRandpretreatmentwithbothAChEinhibitors
Fisher’sposthoctest.Statisticalsignificancewasdefinedas𝑃<0.05.
didnotinfluencetheeffectofinflammationonthisreceptor
gene expression. On the other hand, preceding injection of
DonepezilandNeostigminediminish(𝑃<0.05)suppressory
effectofinflammationontheLH𝛽mRNAexpressioninthe
stimuli,becauseprolongedexpositionofeweontheactionof
AP.NoeffectofanytreatmentonthegeneexpressionofFSH𝛽
bacterialendotoxinwasfoundtodisturbFSHrelease[3,5].It
was determined. It was also determined that LPS injection
isworthmentioningthat,exceptduration,theeffectofendo-
stimulated(𝑃<0.05)geneexpressionofprolactinintheAP,
toxinongonadotropinssecretioncouldbealsodependenton
andneitherDonepezilnorNeostigmineinfluencedthelevel
thecirculatingconcentrationofovariansteroids.Itwasfound
ofprolactinmRNA(Table4).
thatendotoxindelayedthetimetoanexperimentallyinduced
LH surge in ovariectomized ewes but did not alter surge
4.Discussion amplitude,duration,orincidence.ThiseffectofLPSonthe
LHsurgewasdependentuponthemomentwhenendotoxin
The present study showed that peripheral AChE inhibitor, was introduced relative to the onset of the estradiol signal.
Neostigmine, the same as Donepezil, suppressed inhibitory Whenendotoxinwasadministeredearlyintheinitialperiod
effect of acute inflammation on LH release and LH𝛽 gene ofestrogensensitivity,itblockedtheLHsurgeinmostewes,
expression in the AP in ewes during the follicular phase but when endotoxin was administered after the period of
of the estrous cycle. Moreover, in animals treated together estrogensensitivity,thelevelofLHremainedunaffected[26].
with Neostigmine and LPS the circulating level of LH was ThechangesintheendocrineactivityoftheAPseemto
higher than in the control group. The study supports the bearepercussionofeventsoccurringatthelevelofhypotha-
results of our previous experiment on ewes which showed lamus. The study showed that preceding administration of
that systemic AChE inhibitor successfully reduced negative AChE inhibitors reduced the suppressory action of acute
effect of inflammation on LH secretion in the follicular inflammationonGnRHsynthesisinthePOA.However,our
phase ewes [13]. On the other hand, no effect of either results suggest that in the follicular phase of the estrous
AChE inhibitors or acute immune stress was found upon cycle inflammation suppresses the GnRH synthesis at the
thecirculatingconcentrationofFSH.Thisalsosupportsthe posttranscriptionallevel,becausenoeffectofLPStreatment
resultsofpreviousstudiesindicatingthatacuteinflammation onthegeneexpressionofGnRHwasfoundinthehypotha-
didnotinfluencetheFSHreleaseinbothanoestrous[25]and lamic structures containing pericarya of GnRH neurons.
follicular phase ewes [13]. However, other studies on ewes This observation in the follicular phase ewes is generally
showedthatthepotencyofLPStoaffectthesecretionofFSH consistentwiththecharacteristicofGnRHmRNAsynthesis.
may be dependent upon the duration of the inflammatory ThepreviousstudyshowedthattheratioofamountofGnRH
8 BioMedResearchInternational
Table3:Effectoflipopolysaccharide(LPS;400ng/kg;iv.)andacetylcholinesteraseinhibitors:Donepezil(2.5mg/animal;iv.)andNeostigmine
(0.5mg/animal;iv.)injectionsontherelativegeneexpression(mean±SEM;𝑛=6animalspergroup)ofgonadotropin-releasinghormone
(GnRH)inthehypothalamusofewesduringthefollicularphaseoftheestrouscycle.POA:thepreopticarea;AHA:theanteriorhypothalamus;
MBH:themedialbasalhypothalamus;ME:themedianeminence;control:groupinjectedwithsaline;Don.:grouptreatedwithDonepezil;
Neo.:groupinjectedwithNeostigmine;LPS:groupwhichreceivedtheendotoxininjection;Don.+LPS:grouptreatedwithbothDonepezil
andLPS;Neo.+LPS:grouptreatedwithbothNeostigmineandLPS.Inallhypothalamicstructuresgeneexpressiondatawerenormalisedto
theaveragerelativelevelofgeneexpressioninthecontrolewes,whichwassetto1.0.Differentcapitallettersindicatesignificant(𝑃 < 0.05)
differencesaccordingtoatwo-wayANOVAfollowedbyFisher’sposthoctest.
GnRHrelativegeneexpression
Structure
Control Don. Neo. LPS Don.+LPS Neo.+LPS
POA 1±0.1A 0.9±0.1A 0.9±0.2A 0.8±0.2A 0.9±0.1A 1±0.2A
AHA 1±0.1A 1.1±0.1A 0.8±0.2A 0.7±0.1A 0.9±0.1A 0.9±0.1A
MBH 1±0.1A 0.8±0.1A 0.8±0.2A 1.1±0.1A 1±0.1A 1.1±0.2A
ME 1±0.2B 1.3±0.2B 1±0.2B 0.1±0.1A 0.7±0.1B 1.2±0.2B
Table4:Effectoflipopolysaccharide(LPS;400ng/kg;iv.)andacetylcholinesteraseinhibitors:Donepezil(2.5mg/animal;iv.)andNeostigmine
(0.5mg/animal;iv.)injectionsontherelativegeneexpression(mean±SEM;𝑛=6animalspergroup)ofgonadotropin-releasinghormone
receptor(GnRHR),luteinizinghormone𝛽-subunit(LH𝛽),follicle-stimulatinghormone𝛽-subunit(FSH𝛽),andprolactin(PRL)genesin
theanteriorpituitaryofewesduringthefollicularphaseoftheestrouscycle.control:groupinjectedwithsaline;Don.:grouptreatedwith
Donepezil;Neo.:groupinjectedwithNeostigmine;LPS:groupwhichreceivedtheendotoxininjection;Don.+LPS:grouptreatedwithboth
DonepezilandLPS;Neo.+LPS:grouptreatedwithbothNeostigmineandLPS.Thegeneexpressionofeachgenewasnormalisedtothe
averagerelativelevelofgeneexpressioninthecontrolewes,whichwassetto1.0.Differentcapitallettersindicatesignificant(𝑃 < 0.05)
differencesaccordingtoatwo-wayANOVAfollowedbyFisher’sposthoctest.
Anteriorpituitary
Gene
Control Don. Neo. LPS Don.+LPS Neo.+LPS
GnRHR 1±0.1BCD 1.2±0.2BCD 1.4±0.2D 0.5±0.1A 0.9±0.2ABC 0.7±0.2AB
LH𝛽 1±0.1C 0.9±0.1BC 1±0.1C 0.5±0.1A 0.9±0.1BC 0.8±0.1BC
FSH𝛽 1±0.1A 0.8±0.1A 0.8±0.2A 1.1±0.1A 1±0.1A 1.1±0.2A
PRL 1±0.2A 1.1±0.2A 1±0.2A 1.6±0.1B 1.7±0.1B 1.7±0.2B
nuclear mRNA to GnRH cytoplasmic mRNA is 1:2.5 and terminals are located. This observation supports the results
1:1.5,respectively,dependingonthestudy[27,28].Therefore, of previous studies, which showed that immune stress may
a greater amount of nuclear transcript provides a steady reduce the GnRH mRNA transport to the nerve terminals,
flow of GnRH mRNA to the cytoplasm, and it is generally thus reducing the amount of GnRH mRNA in the ME
postulated that changes in the amount of GnRH mRNA [29], but thiseffect maybe restrainedby Rivastigmine[13].
in the perikaryons are rather dependent on this mRNA BecauseitissupposedthatthestorageoftheGnRHmRNA
turnover, both rapid accumulation and fast degradation. It in the nerves terminals may be an element of the system
shouldbenotedthattheeffectofinflammationontheGnRH supporting the secretion of this decapeptide, the ability of
mRNA expression in the hypothalamus of sheep may be AChE inhibitors to restore the amounts of GnRH mRNA
influenced upon the circulating concentrations of estradiol. in the ME could have a profound positive influence on
Thestudyonewesduringanestrousseason,whenthelevelof the GnRH secretion. This may be one of the mechanisms
estradiolispresumablylow,showedthatendotoxin-induced responsiblefortheprotectiveactionoftheAChEinhibitors
inflammationdecreasedthetranscriptionofGnRHmRNAin ontheGnRH/LHsecretionduringanimmune/inflammatory
thePOA[29].Moreover,previousstudyshowedthatcentral challenge.ItwasdescribedthatinflammationdisturbsGnRH
action of potent proinflammatory cytokine, IL-1𝛽, may be release in ovariectomized ewes decreasing GnRH pulse
responsibleforthesuppressionofthetranslationalefficiency amplitudewithoutaffectingtheGnRHpulsefrequency[31],
ofGnRHmRNAintherat[30]andsheep[8]hypothalamus. but our study showed that Rivastigmine not only reduced
Therefore, it seems that in the present study the decrease thesuppressoryeffectofinflammationontheGnRHrelease
found in the content of GnRH in the POA in LPS treated but also even stimulated this neurohormone secretion into
ewesmayresultfromreducedtranslationofthisdecapeptide, thecerebrospinalfluidofewesinthefollicularphaseofthe
and in turn the ability of AChE inhibitors to blockade this estrouscycle[13].
negative effect on the GnRH synthesis may result from the The ability of Neostigmine and Donepezil to block the
suppressionofthelevelofcentralcytokines. effectofinflammationonGnRHsecretioninthehypothala-
In the present study both Neostigmine and Donepezil musmayprimarilyresultfromattenuationtheinflammatory
treatmentcompletelyabolishedLPS-induceddecreaseinthe signal from periphery to the brain parenchyma. As it was
content of GnRH mRNA in the ME, where GnRH nerve mentionedabove,itisconsideredthatthemainmechanism
BioMedResearchInternational 9
via endotoxin-induced inflammation disturbing the GnRH Inthepresentstudy,neitherDonepezilnorNeostigmine
secretioninthehypothalamusisthecentralactionofinflam- treatment influenced the circulating concentration of the
matory cytokines. In our previous study, it was found that stressmarkers:cortisolandprolactinwhichexcludesthatthe
peripheraladministrationofRivastigmineinhibitedtheLPS- effectofAChEinjectionontheGnRH/LHsecretionaswell
induced synthesis of IL-1𝛽 in and gene expression of IL- as GnRHR protein expression results from the attenuation
1 receptors in the hypothalamus of ewe [12]. However, the of stress reaction induced by an immune/inflammatory
actionofRivastigmineaswellasDonepezilissystemic;these challenge.Thestimulatoryeffectofimmuneresponseonthe
compoundsinhibittheAChEactivityandleadtoelevationof releaseofcortisolandprolactinhasbeenpreviouslydescribed
AChconcentrationinboththeperipheralandcentraltissues insheep[13,25,37].Cortisolisconsideredasanimportant
[32].However,theeffectivenessofDonepezilinthepreven- inhibitor of the HPG axis activity, targeting the LH release
tion of inflammatory dependent changes in the GnRH/LH [37].However,thesuppressiveeffectofcortisolontherelease
secretion in the follicular phase does not surprise because of LH release depends on the reproductive status of ewes.
obtained results are concomitant with those described in Theovariansteroids,particularlyestradiol,enablethecortisol
our study with the use of Rivastigmine [13]. The fact that suppressionofLHpulsefrequencyinsheep.Whereascortisol
pretreatment with Neostigmine also abolished suppressive seemstominimallyaffectLHreleaseintheovariectomized
effectofLPStreatmentontheGnRH/LHsecretionsuggests ewes devoid of gonadal steroids [38, 39]. Moreover, it was
that the inhibition of proinflammatory cytokines secretion found that the other components of the HPA axis such
in the peripheral tissues by Neostigmine is sufficient to as CRH and arginine vasopressin may inhibit the pulsatile
block the transition the inflammatory signal into the brain GnRH/LH secretion [40]. Also prolactin may suppress LH
parenchyma,whichwaspreviouslydescribedbyPollaketal. secretion.Itisknownthatthephysiologicalstatesassociated
[11].ThisshowsthatperipheralandsystemicAChEinhibitors withelevatedprolactinbloodconcentration(i.e.,pregnancy,
characterizesimilareffectivenessinthepreventionofinflam- pseudopregnancy,postpartum,andlactation);theLHsecre-
matory dependent distribution of GnRH/LH secretion and tion is always decreased [41]. Circulating prolactin crosses
suggests that pivotal mechanism in the pathophysiology the blood-cerebrospinal fluid barrier and reaches the brain
of neuroendocrine disorders occurring during an immune parenchyma; therefore, the prolactin dependent inhibition
challenge is the elevation of peripheral proinflammatory of LH release could result from its action on the GnRH
cytokinesconcentrationtothelevelnecessarytotransitionof secretion. The results of in vitro studies conducted on the
theinformationabouttheongoingperipheralinflammation GT1neuronalcelllineshowedthatprolactindirectlyinhibits
intothebrain. GnRH release and possibly gene expression in these cells
The proceeding injection of both AChE inhibitors pre- [41].InourpreviousstudyRivastigminetreatmentdecreased
vented inflammatory dependent decrease in the expression the plasma concentration of these hormones in ewes but
ofGnRHRproteinbutdidnotsignificantlyinfluenceonthe not to the control values [13]. However, the background of
GnRHR gene expression. During inflammatory condition, this Rivastigmine action was not completely clear because
reduced expression of GnRHR in the AP may result from ACh is considered to be a stimulant of cortisol [42] and
decreased secretion of the hypothalamic GnRH. It was prolactin[43]release,aswellasthehypothalamus-pituitary-
described that GnRH is one of the most potent regulators adrenal (HPA) axis activator [44]. It was speculated that
ofitsownreceptorexpression.Thisdecapeptideactivatesthe the reduction of cortisol and prolactin release might result
transcriptionalactivityofitsownreceptorgenethroughmul- fromtheanalgesicactionofAChinhibitorsmodulatingthe
2+
tiplepathways,includingcAMP-,PKC-,andCa -dependent inflammatory pain [45] and decreasing the production of
signaltransductionpathways[33].Itisworthmentioningthat proinflammatorycytokineswhicharealsoable to stimulate
theeffectofGnRHontheexpressionofitsownreceptorinthe theactivityoftheHPAaxis[46].Thepresentstudysuggests
APiscloselydependentuponcharacterofitsaction.When thatthestress-reducingeffectofAChEinhibitorsmaybenot
this neurohormone is released in the pulsatile fashion, it universal property of all these compounds or may depend
maintainssteady-stateconcentrationsofGnRHRmRNAand upon used dose of the drug. However, obtained results
numbers of GnRH receptors in the pituitary gonadotropes. support the current view about no pivotal role of cortisol
ButincontrasttotheeffectsofpulsatileGnRH,continuous in the inhibition of the HPG axis during inflammatory
infusionofGnRHleadstoadesensitizationofgonadotropes conditions.Thisobservationisgenerallyconsistentwiththe
andreductioninthenumberofGnRHR[34].However,the study which showed that the activation of the HPA axis is
suppressionofGnRHRgeneexpressionduringinflammation not essential for reproductive disorders during endotoxin-
maybealsocausedbyproinflammatorycytokinesandstress inducedinflammatorychallenges[37].
because it was shown that both IL-1𝛽 and corticotropin- From one hand, it seems that our present study also
releasing hormone (CRH) suppressed GnRHR expression negatively tested our hypothesis formulated in the study
[30, 35, 36]. The fact that preceding injection of AChE with the usage of Rivastigmine claiming that stimulating
inhibitorsdoesnotallowthereductionofGnRHRexpression effectoftheAChEinhibitorontheGnRHsecretionduring
in the AP during an immune/inflammatory challenge may immune stress may result from the accumulation of ACh
have a profound importance for the reactivity of the AP inthebrain.Thisthesiswasjustifiedbecausetheregionsof
because the factor determining ability and strength of the the brain that exhibit cholinergic activity have projections
pituitarygonadotropesresponse to GnRH is the amount of to the POA and therefore may regulate GnRH neuron
GnRHR[34]. activity [47, 48]. Moreover, in vitro experiment performed
10 BioMedResearchInternational
onrathypothalamictissueculturesdemonstratedthatACh References
stimulatedGnRHrelease[49].Aninvitrostudyperformed
[1] P.A.Nepomnaschy,E.Sheiner,G.Mastorakos,andP.C.Arck,
on rat hypothalamic neurons and GT1-7 line cells showed
“Stress,immunefunction,andwomen’sreproduction,”Annals
thatAChmodulatedGnRHreleaseinanenhancedwayand
oftheNewYorkAcademyofSciences,vol.1113,pp.350–364,2007.
actedthroughdifferentcholinergicreceptorsubtypestoexert
[2] M.Caldani,M.Batailler,J.C.Thie´ry,andM.P.Dubois,“LHRH-
stimulatoryandinhibitoryeffects[50].However,inourstudy
immunoreactivestructuresinthesheepbrain,”Histochemistry,
animalstreatedonlywithDonepezilorNeostigminedidnot
vol.89,no.2,pp.129–139,1988.
showanychangesintheGnRH/LHsecretionwhichsuggests
[3] D. Tomaszewska-Zaremba, A. Herman, and K. Haziak,
that maintaining of undisturbed GnRH/LH secretion in
“How does bacterial endotoxin influence gonadoliberin/
animals concomitant treated with LPS rather results from
gonadotropins secretion and action?” Journal of Animal and
the anti-inflammatory action of this AChE inhibitors than
FeedSciences,vol.25,no.4,pp.283–291,2016.
simplyfromaccumulationofAChinthehypothalamus.On
[4] A.P.Herman,K.Kopycin´ska,A.Krawczyn´ska,K.Romanowicz,
the other hand, the reactivity of the brain tissues during
andD.Tomaszewska-Zaremba,“Theeffectofrepeatedendo-
inflammatoryconditionmaybechanged.Therefore,certainly toxininjectionsongonadotropinsecretioninewes,”Journalof
itcannotbestatedthatAChdoesnotplaysomeroleinthe AnimalandFeedSciences,vol.23,no.3,pp.217–221,2014.
AChE inhibitors action on the secretion of GnRH and/or [5] D. F. Battaglia, H. B. Krasa, V. Padmanabhan, C. Viguie´, and
LH during inflammation because in this physiological state F. J. Karsch, “Endocrine alterations that underlie endotoxin-
itmayinfluencetheresponsivenessofboththehypothalamic induceddisruptionofthefollicularphaseinewes,”Biologyof
and AP tissues on the ACh action. It was previously found Reproduction,vol.62,no.1,pp.45–53,2000.
thatLPSinfluencestheprofileofAChreceptorswhichmay [6] H.WatanobeandY.Hayakawa,“Hypothalamicinterleukin-1𝛽
change responsiveness of the cells on the action of this andtumornecrosisfactor-𝛼,butnotinterleukin-6,mediatethe
neurotransmitter[51].Inourstudywedeterminedthatinani- endotoxin-inducedsuppressionofthereproductiveaxisinrats,”
malstreatedwithNeostigmineandLPSthemeancirculating Endocrinology,vol.144,no.11,pp.4868–4875,2003.
concentrationofLHwashigherthaninthecontrolgroup,but [7] C. Rivier and W. Vale, “Cytokines act within the brain to
inhibitluteinizinghormonesecretionandovulationintherat,”
thiseffectwasnotparalleltothechangesintheGnRHcontent
Endocrinology,vol.127,no.2,pp.849–856,1990.
inthePOA.ThissuggeststhattheLHsecretionisenhanced
[8] A.P.Herman,T.Misztal,K.Romanowicz,andD.Tomaszewska-
bytheperipheralfactorsreachingdirectlytheAP.Although
Zaremba,“CentralinjectionofexogenousIL-1𝛽inthecontrol
the ex vivo study suggested that stimulatory action of ACh
activities of hypothalamic-pituitary-gonadal axis in anestrous
ontheLHsecretionfromtheAPisindirectandistargetedon
ewes,”ReproductioninDomesticAnimals,vol.47,no.1,pp.44–
thestimulationofthehypothalamicGnRHrelease[52],more
52,2012.
presentstudyshowedthattheAChreceptorsarepresentand
[9] M.Rosas-Ballina,M.Ochani,W.R.Parrishetal.,“Splenicnerve
active in the majority of AP cells [53]. Therefore, it cannot
is required for cholinergic antiinflammatory pathway control
be excluded that at least partially the stimulatory effect of
ofTNFinendotoxemia,”ProceedingsoftheNationalAcademy
Neostigmine and LPS treatment on the LH secretion may ofSciencesoftheUnitedStatesofAmerica,vol.105,no.31,pp.
result from the accumulation of ACh in the blood which 11008–11013,2008.
directlyaffectsthesecretoryactivityofAP. [10] L. V. Borovikova, S. Ivanova, M. Zhang et al., “Vagus nerve
Insummary,ourstudyshowedthatperipheralinhibitor stimulationattenuatesthesystemicinflammatoryresponseto
of AChE activity, Neostigmine, effectively abolished the endotoxin,”Nature,vol.405,no.6785,pp.458–462,2000.
suppressive effect of acute inflammation on the GnRH/LH [11] Y.Pollak,A.Gilboa,O.Ben-Menachem,T.Ben-Hur,H.Soreq,
secretionandthiseffectwasgenerallysimilartothesystemic and R. Yirmiya, “Acetylcholinesterase inhibitors reduce brain
actionofDonepezil.Thisindicatesthatinflammatorydepen- andbloodinterleukin-1𝛽production,”AnnalsofNeurology,vol.
dentchangesintheGnRH/LHsecretionmaybeeliminated 57,no.5,pp.741–745,2005.
or reduced by the compounds suppressing inflammatory [12] A. P. Herman, A. Krawczyn´ska, J. Bochenek et al., “Inhibi-
reactiononlyattheperipherywithouttheneedforinterfering tionofacetylcholinesteraseactivitybyrivastigminedecreases
lipopolysaccharide-inducedIL-1𝛽expressioninthehypothala-
in the CNS. Our study suggests that AChE inhibitors not
musofewes,”DomesticAnimalEndocrinology,vol.44,no.3,pp.
capableofcrossingtheBBBmightpotentiallybeusedinthe
109–114,2013.
therapyofinflammatoryinducedneuroendocrinedisorders.
[13] A.P.Herman,A.Krawczyn´ska,J.Bocheneketal.,“Theeffect
ofrivastigmineontheLPS-inducedsuppressionofGnRH/LH
ConflictsofInterest secretion during the follicular phase of the estrous cycle in
ewes,”AnimalReproductionScience,vol.138,no.3-4,pp.203–
Alloftheauthorshavedeclaredthattherearenoconflictsof 212,2013.
interestregardingthiswork. [14] A.Russel,“BodyConditionScoringofSheep,”inSheepandGoat
Practice,E.Boden,Ed.,BailliereTindall,Philadelphia,Pa,USA,
1991.
Acknowledgments [15] R.R.Ro´s,Ed.,NutrientRequirementsforCattleAndSheepin
TheTraditionalSystem,InstytutZootechniki,Krakow,Poland,
This research was supported by the funds granted by 1993.
the National Science Centre based on Decision no. DEC- [16] A.P.Herman,A.Krawczyn´ska,J.Bochenek,H.Antushevich,A.
2013/11/B/NZ9/01848. Herman,andD.Tomaszewska-Zaremba,“Peripheralinjection