Background

It is well established that immune stress caused by infections and inflammatory diseases reduces animals productivity and is a powerful modulator of mechanisms regulating reproduction at all levels of the hypothalamic-pituitary-gonadal axis[1].A few pathways have been suggested to be responsible for the immunemediated inhibition of reproductive activity at the level ofhypothalamusand　one　oftheseinvolvesproinflammatory mediators such as cytokines[2].Hypothalamic interleukin　(IL)-1β and tumor necrosis factor(TNF)-α mediates the lipopolysaccharide(LPS)-induced suppression of gonadotropin releasing hormone(GnRH)and luteinizing hormone(LH)release in female rats[3].In ewes,the central IL-1β is an important modulator of the GnRH biosynthesis and release during immune/inflammatory challenge.The thesis about the crucial role of these cytokines in the transmission of signals from the immune to neuroendocrine systems seems to be supported by the presence of IL-1β and its receptors in the hypothalamus in LPS-treated ewes[4].

In general,passage of molecules from the periphery to the brain is restricted by brain barriers:blood-brain barrier(BBB)located in cerebromicrovascular endothelial cells and blood-cerebrospinal fluid barrier(BCSFB)located in the epithelial cells of choroid plexus(CP)[5].The origin of central pro-inflammatory cytokines is differentiated.At the beginning of inflammation,the main source of cytokines present in the cerebrospinal fluid(CSF)is peripheral circulation,while over time of inflammation,endogenous cytokines produced in the brain seems to be more important.In rats treated intravenously with IL-1β two waves of cellular activation at the brain appears,the first one at the blood side of the BBB 30 min after IL-1β administration and then after 3 h at the parenchymal side of the BBB[6].It has been demonstrated in rats,that early after(5 h)injection of IL-1β to the brain BBB becomes permeable to intravenously administered contrast[7].In this process matrix metalloproteinases(MMPs)asenzymesthatcatalyzethe proteolytic cleavage of basal lamina components and thus remodeling of the extracellular matrix and brain barriers permeability play an important role[8].Interestingly,melatonin attenuated BBB hyperpermeability in IL-1β stimulated rat brain microvessels endothelial cells in vitro as well as in vivo in mouse traumatic brain injury model[9].From the periphery,IL-1β is transported throughout the brain barriers[10].Transport of IL-1β has been suggested to occur via a type II IL-1 receptor[11].This receptor may also be released from cells and function as decoy receptor to block IL-1β action in contrast to type I IL-1β receptor that transduce IL-1 signals after binding with IL-1β[12,13].Expression of type II IL-1 receptor mRNA(Il1r2)and I IL-1β receptor(Il1r1)was detected in the brain endothelial cells[14]and in the CP[15].In ewes,intravenous injection of LPS is one model of systemic inflammation which has been used to study mechanisms responsible for the immune-mediated inhibition of reproductive activity[4].So far little is known about CSF concentration of IL-1β in ewes and its correlation　with　plasmaconcentration　duringLPS-induced systemic inflammation.Melatonin receptors MT1 and MT2 has been demonstrated in the ovine CP what unable direct melatonin action on the CP[16].Melatonin action on the concentration of IL-1β in blood plasma and the CSF is particularly interesting due to the use of melatonin from continuous slow-release implants to advance the onset of a breeding season in sheep and goats[17].

The present study aimed at evaluating effect of LPS alone and with melatonin slow-release implants on the concentration of IL-1β in blood plasma and the CSF as well as on the BBB permeability in ewes early after LPS administration.Additionally,we　evaluated　effectof melatonin implantation on mRNA expression of Il1B and its receptors Il1r1 and Il1r2 as well as Mmp3 and Mmp9 in the CP under the influence of IL-1β.

Methods

Animals and experimental design

All animal procedures were conducted in accordance with the Polish Guide for the Care and Use of Animals and approved by the Local Ethics Committee(No.25/2012).Female adult sheep(4-5 years old,50-60 kg body weight)of the Blackheaded Mutton breed(n=14)were maintained indoors under natural lighting conditions(latitude 52°N,21°E)and fed a constant diet of hay,straw and commercial concentrates according to the recommendations proposed by the National Research Institute of Animal Production for adult ewes.Water and mineral licks were available ad libitum.On the beginning of May,ewes were ovariectomized under general anaesthesia　and　then　(middle　ofMay)subcutaneously implanted with an oestradiol(E2)implant,which maintained plasma E2 concentrations of 2-4 pg/L[18].After 2 wk of recovery ewes were implanted under general anaesthesia with stainless steel guide cannulae(1.2 mm o.d.)into the third ventricle as described earlier[19].In the middle of May,ewes were first sampled for blood and then one-half of the animals was randomly melatonin-implanted(n=7,slow-release implant of 18 mg,Melovine Ceva Sante Animale,France)and the second half of the animals was sham-implanted(n=7).Approximately 40 d later,ewes were implanted with the jugular vein catheter early on the morning(7:00 am)and have been placed in individual cages where they could lie down and have access to hay and water.To prevent the stress of social isolation,all ewes had visual contact.After that stainless catheter was introduced into the third ventricle and control blood and CSF samples were collected.Then immune stress was induced by intravenously injection of LPS from Escherichia coli 055:B5(Sigma,USA),at the dose of 400 ng/kg of body weight,dissolved in saline(0.9%w/v NaCl)at a concentration of 10 mg/L(10 μg/mL)as it was used previously in ewes[20].The individual body mass of experimental ewes were at the range of 52 kg to 63 kg,therefore injection volume of LPS solution/saline was at the range of 2.1 to 2.5 mL.Body temperature was measured before and after LPS administration.Blood plasma was collected just before and after melatonin implantation and then every hour after LPS administration(Fig.1).Blood samples were collected through a catheter inserted into the jugular vein.First 2 mL of blood samples were removed then 10 mL were collected into the tubes with stabilizer(EDTA).Immediately　aftercentrifuging,the　blood plasma were divided into separate(1 mL)aliquots and stored at-40 °C until assayed for IL-1β and albumin.To collect the CSF samples,a stainless steel catheter(1.0 mm o.d.,0.8 mm i.d.)was carefully introduced into the guide cannula and connected to a special cannula-Eppendorf tube system joined to a PHD 2000 infuse/withdrawal pump(Hugo Sachs Elektronik Harvard Apparatus,Germany).The CSF collection from the third ventricle of conscious ewes was performed during a 7 h period(1 h before and 6 h after LPS administration)at a rate of 20 μL/min.The tubes of CSF samples were kept in an ice bath during sampling,and the volume of one sample collected during the 30 min period was about 500 μL.Immediately after filling,the tubes were stored at-80 °C until assayed for IL-1β and albumin.The ewes were euthanized 6 h after LPS administration.After decapitation,the brains were dissected,CP were removed from their anchoring to the Galien’s vein and the split was made along the mid-line,separating the CP from each lateral ventricle.CPs were then immediately frozen in liquid nitrogen and stored at-80°C until use.

Hormones and IL-1β concentration measurement

此后，张纯如回到中国，走访大屠杀幸存者及当年的施暴者，花3年时间，写就此书。采访中，张纯如始终不解，为何会有一个日本兵屠杀一村人的怪事？甚至，几百名被日军俘虏的军人面对仇敌，居然如待宰的羔羊，眼看着同伴被拉出去行刑。1997年，当《南京暴行：被遗忘的大屠杀》出版，被西方媒体誉为第一部全面记录日军对南京所犯暴行的英文著作时，张纯如心绪难平。

Western blotting and CSF/blood plasma quotient

他从网上调出了一个网页，上面写着“公众接遇赔偿费制度”。这项制度是在1963年开始实施的，距今已有半个多世纪的历史了。

The ability of the melatonin implants to maintain permanently high blood concentrations of melatonin was monitored by determining melatonin concentrations in blood plasma samples obtained before and 40 d after melatonin-implantation using radioimmunoassay(RIA)described by Misztal et al.[21].The assay sensitivity for melatonin was 16.8±8.0 pg/mL,and the intra-and interassay coefficients of variations were 10.5 and 13.2%,respectively.Blood plasma and CSF IL-1β concentrations were determined by commercially available enzymelinked immunosorbent assay(ELISA Kit for ovine Interleukin 1β,Cloud-Clone Corp.,USA),following the manufacturer’s instructions.The optical density of individual wells was measured by a spectrophotometric microplate reader(Epoch,BioTek,Switzerland)at a wavelength of 450± 10 nm.The concentration of IL-1β in samples were determined by comparing the optical density of the samples to the standard curve.The detection limit of the assay was less than 5.9 pg/mL.

Fig.1　Schematic diagram of the experimental design.At the beginning of May all the ewes(n=14)were ovariectomized and implanted with an oestradiol(E2).Two weeks later,animals were implanted under general anaesthesia with stainless steel guide cannulae into the third ventricle and ewes were melatonin-(n=7)or sham-implanted(n=7).Approximately 40 d later,melatonin-and sham-implanted ewes were treated with lipopolysaccharide(LPS).Blood samples were collected for melatonin,cortisol albumin and IL-1β concentrations measurement.Cerebrospinal fluid(CSF)samples were collected for albumin and IL-1β concentrations measurement.The choroid plexuses were collected 6 h after LPS administration

Statistical analysis

Abbreviations

ThetotalRNA　from　theCPwasisolatedusing NucleoSpin RNA II Kit(Marcherey-Nagel,Germany).All steps of the isolation were performed according to the manufacturer’s protocol.The purity and concentration of the isolated RNA were quantified spectrophotometrically using a NanoDrop 1000 instrument(Thermo　FisherScientific,USA).Theintegrityof RNA was verified by electrophoresis using 1.2%agarose gel stained with ethidium bromide(Sigma Aldrich,USA).TosynthesizecDNA,theDyNAmo cDNA Synthesis Kit(Thermo Fisher Scientific,USA)and 1 μg of total RNA were used.Expression of interleukin 1β(Il1B)and its receptor type I(Il1r1)and　typeII(Il1r2)and　matrix　metalloproteinases(Mmp)3 and 9 in the ovine CP was determined by real-time PCR.Specific primer pairs for the different genes were used according to the literature or were designed using Primer-BLAST(National Center for Biotechnology Information)and were synthesized by Genomed(Poland)and are presented in Table 1.One reaction mixture for real-time PCR reaction(10 μL)contained 3 μL of diluted(1:14 reference genes,1:10 Il1B,Il1r1,Il1r2 and 1:8 Mmp3 and Mmp9)cDNA,0.2 μmol/L of the forward and reverse primers and 5 μL of mastermix from a DyNAmo SYBR Green qPCR Kit with ROX(Thermo Fisher Scientific,USA).The following protocol was used:95°C for 10 min for Hot Start modified Tbr DNA polymerase,followed by 35 cycles of 15 s of denaturation at 95°C,30 s of annealing at X°C(see Table 1)and 30 s of extension at 72°C.After the cycles,a final melting curve analysis under continuous fluorescence measurement was performed to evaluate the specific amplification.The results were analyzed using Real-time PCR Miner(on-line available:http://www.miner.ewindup.info/version2),based on the algorithm developed by Zhao and Fernald[22].

Table 1　Sequences of oligonucleotide primers used for real time-PCR

GenBank Acc.No.　Gene　Amplicon size,bp Temp.of primers annealing,°C Forward/reverse Sequence 5′→ 3′Reference X54796.1　Il1B　137　59　forward　CAGCCGTGCAGTCAGTAAAA　]reverse　GAAGCTCATGCAGAACACCA NM_001206735.1　Il1r1　124　59　forward　GGGAAGGGTCCACCTGTAAC　]reverse　ACAATGCTTTCCCCAACGTA NM_001046210　Il1r2　161　59　forward　CGCCAGGCATACTCAGAAA　]reverse　GAGAACGTGGCAGCTTCTTT XM_004015970.1　Mmp3　112　60　forward　AAGGCAGACATTTTTGGCGG　Originally designed reverse　ATGCCTCTTGGGGAACCTGC XM_004014614.1　Mmp9　115　60　forward　CTTCCGATGGAAAGAACGGGC　Originally designed reverse　GGGATCACAACGCCTTTGC　Gapdh　143　60　forward　TGACCCCTTCATTGACCTTC　]reverse　GATCTCGCTCCTGGAAGATG NM_001009784.1　Actb　122　60　forward　GCCAACCGTGAGAAGATGAC　]reverse　TCCATCACGATGCCAGTG BC_108088.1　Hdac1　115　60　forward　CTGGGGACCTACGGGATATT　]reverse　GACATGACCGGCTTGAAAAT

Fig.2　The mean(±SEM)body temperature before and every 1 h for 5 h after LPS treatment in sham-implanted(solid line)and melatonin-implanted(dotted line)adult ewes

To assess the functions of BCSFB albumin quotient(QAlb)was used.The CSF and blood plasma samples(equal volume 16 μL CSF and diluted blood plasma(1:160)with addition of 4 μL of loading buffer)from the sham-and melatonin-implanted group(one animal from each group)were loaded onto 10%sodium dodecyl sulfate(SDS)polyacrylamide gels together with serial dilutions of sheep serum albumin(SSA,Biorbyt,USA,range 0.25 to 5 μg/μL).Electrophoresis was performed using　theMiniProtean　IIelectrophoreticapparatus(BioRad,USA)at 60 mV constant voltage.Thereafter,proteins were transferred onto 0.2 μm thick nitrocellulose membranes(Whatman Inc.,Germany)at 30 V for 1.5 h in a semi-dry transfer system(BioRad,USA).After 1.5 h blocking with block buffer(TBST,50 g/L nonfat milk in 10 mL Tris buffer saline containing 0.5%Tween 20)at room temperature,the membranes were extensively washed in TBST and incubated overnight at 4°C with rabbit polyclonal antibodies against sheep albumin at 1:300 dilution(Anitbodies-online,Germany).After final wash,membranes were developed using chemiluminescence SuperSignal®West Dura Kit(Thermo Scientific,USA)andvisualizedbyVersaDoc4000MP Imaging System(BioRad,USA).Based on a SSA dilution curve,the albumin concentration was calculated in both CSF and blood plasma samples by measuring optical densities of the bands(Image Lab 5.2.1,Software,BioRad,USA).The integrity of BCSFB was estimated by the ratio of albumin concentrations in CSF and blood plasma.The albumin quotient was evaluate as follows:QAlb=Alb(CSF)/Alb(blood plasma).

All data are presented as the mean±standard error of the mean[SEM].The real-time PCR results are presented as the relative gene expression of the target gene vs.the mean of 3 reference genes(Gapdh,Actb,Hdac1).The body temperature,melatonin and IL-1β concentrations,were analyzed by a one-way ANOVA for repeated measures and relative gene expression by t-test using PRISM 6 GraphPad Software(San Diego,USA).For statistical analysis,percentage data of QAlb were multiplied by 0.1 and then arcsin transformed according to the eq.(Y=deg.(arcsin(sqrt(Y/100)))using PRISM 6 GraphPad Software.The transformed data were subjected to oneway ANOVA.Additionally,the differences between QAlb in sham-and melatonin-implanted ewes before LPS administration were analyzed by Welch test.The relationship between variables was analyzed using Pearson’s correlation coefficient.Statistical significance was assumed at P＜0.05.

Results

The mean body temperature before the LPS administration was 39.6± 0.1°C in the sham-implanted group and 39.9 ± 0.2 °C in the melatonin-implanted group and increased significantly(P≤0.05)to 41.6±0.1°C and 41.7± 0.1°C 4 h after LPS administration,respectively(Fig.2).Additionally,in all ewes LPS administration induced rapid breathing and shortness of breath,sneezing,and stopped feed intake,anhedonia and reduced social interactions which are collectively termed‘sickness behavior’.The plasma melatonin concentration in melatoninimplanted ewes increased significantly(P＜0.05)from 8.2±2.2 pg/mL(before implantation)to 84.6±15.0 pg/mL　(onemo　afterimplantation)whilein　shamimplanted stayed on the level of 9.6±1.1 pg/mL.As shown on Table 2 immunoreactive IL-1β was not detected in blood plasma collected 1 h before LPS in 2 out of 6 ewes in sham-implanted group and in 4 out of 7 ewes in melatonin-implanted group.In other ewes IL-1β concentration ranged from 49.4 to 197.2 pg/mL and 128.6 to 192.7 pg/mL in sham-and melatonin-implanted ewes,respectively.Two and a half h after LPS administration the concentration of IL-1β increased in all investigated　animals　and　reached　the　mean　levelof 159.3±53.1 pg/mL and 197.8±42.8 pg/mL in shamand melatonin-implanted ewes,respectively.Melatonin did not affect IL-1β concentration in blood plasma.In CSF collected before LPS,IL-1β was not detected in 2 out of 7 ewes in melatonin group.In sham-implanted ewes IL-1β ranged from 13.5 to 51.7 pg/mL while in other melatonin-implanted ewes ranged from 11.8 to 67.4 pg/mL.After LPS treatment the mean concentration of IL-1β increased in all investigated animals in sham-implanted(129.8±54.2 pg/mL)and melatoninimplanted(139.6±51.5 pg/mL)ewes.There was no effect(P ＞0.05)of melatonin on CSF IL-1β concentration as well as no differences(P ＞ 0.05)between IL-1β inblood plasma and CSF after LPS administration in both groups.No correlations was found between plasma and CSF IL-1β concentration after LPS administration in both sham-and melatonin-implanted groups(r2=0.08;P＜0.29 vs.r2=0.01;P＜0.4).

Table 2　Individual measurements of IL-1β concentration(pg/mL)in blood plasma and cerebrospinal fluid(CSF)in sham-implanted and melatonin implanted ewes before and 3 h after lipopolysaccharide(LPS,400 ng/kg)administration

asignificant vs.concentration before LPS administration at P＜0.05

Sham-implanted Melatonin-impla Plasma,pg/mL　CSF,pg/mL　Pl Ewe#　Before　After LPS　Before　After LPS　Ewe#　Be 1 0.0　65.3　27.8　48.0　1　14 2 0.0　105.9　13.5　35.5　2　12 nted asma,pg/mL　CSF,pg/mL fore　After LPS　Before　After LPS 3.3　211.0　26.4　410.8 8.　134.7　67.4　146.5 3 53.0　58.5　21.0　50.4　3　0.0　210.7　19,2　39.6 4 76.1　113.0　20.3　365.6　4　0.0　298.4　0.0　198.5 5 197.2　399.0　22.8　66.7　5　0.0　80.3　0.0　12.3 6 49.4　214.2　51.7　213.7　6　192.7　378.7　24.2　54.0 7 0.0　71.0　11,8　115.4 Mean(±SEM)62.0(±29.7)159.3a(±53.1)26.2(±5.4)129.8a(±54.2)66.4(±32.1)197.8a(±42.8)21.3(±8.7)139.6a(±51.5)

Fig.3　Determination of albumin levels by western blot analysis in sheep cerebrospinal fluid(csf)and blood plasma samples(bp)collected before and 6 h after LPS administration.The densities of the bands were based on sheep serum albumin(SSA)dilution curve

The albumin concentrations in the CSF collected from the third brain ventricle and blood plasma were calculated on the base of linear dilution curve of sheep serum albumin detected by western blot method(Fig.3)and then the integrity of BCSFB was estimated by ratio of albumin concentrations in the CSF and blood plasma(QAlb).Six hours after LPS administration mean QAlb was on the level of 0.20%±0.05 and 0.14%±0.02(P＞0.05)in sham-and melatonin-implanted ewes and was similar to that observed before LPS administration(0.18%±0.02 vs.0.12%±0.03,P=0.0572,Fig.4).mRNA expression of Il1B and its receptors Il1r1 and Il1r2 in the CP collected 6 h after LPS administration were similar(P＞0.05)in both sham-and melatoninimplanted group(Fig.5).Within 35 amplification cycles mRNAexpressionofMmp3andMmp9wasnot detected.

Discussion

Fig.4　Mean(±SEM)cerebrospinal fluid albumin quotient(QAlb)before and after lipopolysaccharide(LPS,400 ng/kg)administration in sham-(circles)and melatonin-implanted(squares)adult ewes

Our results show that in ovariectomized and E2 treated ewes LPS in a dose of 400 ng/mL increased IL-1β concentration in blood plasma and the CSF early(2.5 h)after LPS administration.In our previous study LH secretion as well as the GnRH release was observed to be suppressed 3 h after intravenous LPS administration in ewes[23].The peripheral administration of LPS increases pro-inflammatory cytokine level IL-1β,TNFα and IL-6 in blood plasma in many animals,however,there are species differences in a dose of LPS necessary to trigger the response[24-26].Ewes are much more sensitive to LPS in comparison to mice and rats in which the range of the applied LPS doses is from 5 μg/kg to 5 mg/kg.In our study the mean concentration of IL-1β reach the level of 159.3±53.1 pg/mL in plasma and 129.8±54.2pg/mLinCSFincontroland 197.8±42.8 pg/mL in plasma and 139.6±51.5 pg/mL in CSF of melatonin-implanted ewes.Individual plasma IL-1β levels were differentiated in both groups but means were similar to these observed in pigs 3 h after continuous LPS infusion in a dose of 250 ng/kg/h[26].We did not find any significant differences in IL-1β mean　concentration　between　groups　(sham-　and melatonin-implanted)as well as between compartments(blood plasma and the CSF).This indicates the lack of melatonin slow-release implants effect on IL-1β concentration in both plasma and CSF early(2.5 h)after LPS administration.In our study,daytime plasma melatonin concentration in melatonin-implanted ewes were similar to this observed in our previous study[27]and reported by Skinner and Malpaux[28]for plasma collected at night.We did not observed any correlation between plasma and CSF concentration of IL-1β in both groups,what may suggests that transport of IL-1β from blood to the CSF is not only the source of IL-1β in the CSF early after LPS administration,and that CSF IL-1β originates from other sources.These findings are in line with results described by Qann and colleagues[29],who observed that in rats subseptic doses of LPS(0.01-10 μg/kg)that are in range of the dose used in ewes induced Il1B mRNA expression only in the CP,the circumventricular organs and meninges.Moreover,secretion of cytokines to the brain by activated cells of the BBB has been described as an additional,to saturable transport system,pathway of cytokine access to the brain[10].

Fig.5　The effect of sham-(white)and melatonin-implantation(grey)on the mean(±SEM)relative relative gene expression for interleukin 1β(Il1B;a)and its receptor type I(Il1r1;b)and type II(Il1r2;c)in the choroid plexus of lipopolysaccharide(LPS)-treated adult ewes.Data presented on each panel were normalized to the average relative quantity of target gene expression in sham-and melatonin-implanted,which was set to 1.0

In addition to evaluating the effect of LPS alone and with melatonin from slow-release implants on the concentration of IL-1β in blood plasma and the CSF,the second aim of this study was to investigate the BBB permeability in ewes early after LPS administration.The integrity of brain barriers was estimated by the ratio of albumin concentrations in CSF and blood plasma.The QAlb,calculated in our study before LPS administration was similar to that obtained by Chen et al.[30]for young(1-2 yr)and middle aged(3-5 yr)ewes.Moreover,the QAlb on the level of 0.18%±0.02 in shamimplanted and 0.12%±0.03 in melatonin-implanted ewes observed in ewes before LPS administration,despite the difference at the very edge of significance(P=0.0572),seem to confirm previous observation related with melatonin as possible modulator of molecule passage throughout the brain barriers in sheep.These include:1)higher steroids access to the CSF during long than short days in female sheep[18,31],2)higher passage of leptin from the periphery to the CSF in rams during long days than short days[32],3)higher expression of tight junction proteins in the CP in ewes during short than long days[33]and 4)photoperiod-dependent change in CSF proteome composition in ewes[34].The lack of differences in QAlb before and after LPS administration,found in our studies,indicates that despite the high level of IL-1β in blood and the CSF the integrity of BBB and BCSFB was not damaged 6 h after LPS administration.Indeed,in the CP collected 6 h after LPS administration the expression of mRNA for Mmp3 and 9,that are responsible for degradation of extracellular matrix and therefore for increase of BCSFB permeability was very weak.

Conclusions

In summary we demonstrated that intravenous LPS administration in ewes induces rapid increase of IL-1β in blood plasma and the CSF that is not modulated by melatonin from slow-release implants.The lack of changes in the brain barrier permeability early after LPS administration,at the time when LPS-dependent suppression of GnRH secretion was observed in ewe,indicates that LPS acts mainly at the BBB and BCSFB which are a place for elaboration of signal molecules that communicate peripheral immune status to the brain.

出站的路上，几位火车站的保安大叔半开玩笑的关怀，一下就让人感受到了天津人的热情，似乎身上也暖和了许多！再往前走，一个开出租车的女司机，看我穿得清凉，主动要给我外套，我说不要紧，一会儿上车就暖和了，她还一个劲儿地要我赶紧穿上，而关键问题是我并不是她的客户。最后我还是固执地没有接受她的外套，只好道声“感谢”！

Relative gene expression assays

在设备列车之间用快速电缆插接，维护和安装好列车内部的管路电缆，系统采用大量的自动开启变频泵来调节压力，平均开放数量比传统的开泵人工控制能够提高20%的效率，而且同时保证工业流量，自动减少开放数量，降低无功损耗。

SJ,HAP and JM conceived and designed the experiment;SJ,KM,SA,MT,JM,HAP and ZK performed animal experiment;KM,SA,MA and ZK performed laboratory analysis;SJ,KM and MA analyzed data;SJ,MK and MT prepared the manuscript.All authors read and approved the final manuscript.

Acknowledgements

We would like to thank Dr.Przemysław Gilun and Joanna Winnicka for expert technical assistance.Marta Kowalewska participated as a part of PhD thesis.

Funding

2.4 温度条件的选择 选择在室温下静置40 min、50 ℃温育40 min、76 ℃温育40 min的3种试验效果对比分析，结果显示，在76 ℃温育40 min下OA、DTX1、DTX2加标回收率最高，而且能够有效地去除一些杂质基质的干扰。因此该试验选择76 ℃温育40 min作为温度条件。

This work was supported by a project funded by the National Science Centre allocated on the basis of decision-DEC 2011/03/B/NZ9/00118.Participation of Dr.T.Misztal,Dr.M.Jalynski and Dr.K.Zabek was supported by Ministry of Science and High Education.

Availability of data and materials

All data supporting our findings are included in the manuscript.

Authors’contributions

Actb:β-actin;BBB:Blood-brain barrier;BCSFB:Brain-cerebrospinal fluid barrier;bp:Blood plasma;CP:Choroid plexus;CSF:Cerebrospinal fluid;E2:Oestradiol;Gapdh:Glycer-aldehyde-3-phosphate dehydro-genase;GnRH:Gonadotropin releasing hormone;Hdac1:Histone deacetylase 1;IL:Interleukin;IL-1R1:Interleukin 1 receptor type I;IL-1R2:Interleukin 1 receptor type II;LH:Luteinizing hormone;LPS:Lipopolysaccharide;Mmp:Matrix metalloproteinase;MT:Melatonin receptor;QAlb:Albumin quotient;RIA:Radioimmunoassay;SDS:Sodium dodecyl sulfate;SEM:Standard error of the mean;SSA:Sheep serum albumin;TBST:Tris buffer saline with Tween;TNF:Tumor necrosis factor

Ethics approval and concent to participate

Experimental protocol was approved by the Local Ethics Committee(No.25/2012).

Consent for publication

《杂文月刊》文摘版2018年10月下刊登的《人走“查”不凉退休并不意味着“软着陆”》一文，给退休官员敲响了警钟，值得深思。

All authors read and approved the final manuscript for publication.

Competing interests

The authors declare that they have no competing interests.

杨春梅和郑岩在研究我国促进技术创新的相关财税政策中发现，当前，我国的财税政策之间缺乏联系，各个税种之间内在联系不紧密，一些新出台的政策和旧政策之间的衔接也不紧密、协调性差[9]。如中央和地方之间财税激励政策之间衔接的不紧密，上下不统一问题。如中发〔2015〕8号和国发〔2016〕16号等“新政”中提出取消科技成果“审批或备案”环节，给予企业、科研院所等更多自主权。但一系配套制度并未做出相应调整，导致财税激励政策上下联动受限，也给企业申报设置了不小障碍。

Author details

2000年，小布什在美国总统大选期间大骂《纽约时报》记者为“白痴”。他骂道：“真是白痴。”随即，小布什的竞选搭档切尼附和说：“没错，而且是第一！”两人都不知，当时麦克风没关。

1Institute of Animal Reproduction and Food Research,Polish Academy of Sciences,Olsztyn,Poland.2The Kielanowski Institute of Animal Physiology and Nutrition,Polish Academy of Sciences,Jablonna n/Warsaw,Olsztyn,Poland.3Veterinary Medicine Faculty,University of Warmia and Mazury,Olsztyn,Poland.4Department of Sheep and Goat Breeding,Animal BioEngineering Faculty,University of Warmia and Mazury in Olsztyn,Olsztyn,Poland.

在转基因小鼠中，研究小组让大脑中的每一个突触在荧光灯下发出光亮，就像繁星点点的夜空一样。正如每颗恒星各不相同，研究小组发现突触也有着很大的差异，但是这种差异显著体现在对记忆和思考的支持作用上。

其次，应当确保日常加强对于生猪健康状况的检查，确保发病的猪群可以得到及时发现，并选用敏感性药物进行对症治疗。在治疗的过程中，除需依照治疗标准确保及早、足量、按疗程进行治理外，还应当考虑到细菌与病菌所可能产生的抗药性特点，针对预防药物进行定期轮换使用，并针对病猪进行药敏试验，以确保选取的药物具备治疗功效。

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