促红细胞生成素治疗宫内感染致新生大鼠脑白质损伤的研究
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摘要
目的：
早产儿脑白质损伤(white matter damage，WMD)被认为是脑瘫发生的最主要的危险因素，给家庭和社会带来了巨大的负担。目前，早产儿WMD的机制尚不十分清楚，流行病学和动物实验研究均表明宫内感染在早产儿WMD的发生机制中起重要作用。WMD的病理特点主要是脑白质的凝固性坏死，坏死部位出现反应性星形胶质化、少突胶质细胞的丢失、髓鞘损伤和轴突损害。近年来研究认为，WMD的发生与脑内细胞因子的关系非常密切。细胞因子能以多种机制导致脑白质损伤：1．损伤少突胶质细胞、星形胶质细胞、髓鞘和轴突等；2．通过诱导其他细胞因子而进一步产生细胞损害；3．介导一氧化氮(nitric oxide，NO)的神经毒性作用等。官内感染导致早产儿WMD的机制可以推测为由细胞因子介导的一系列脑组织中各种细胞和蛋白合成的变化所致。
近年来的研究发现促红细胞生成素(erythropoietin，EPO)在脑和其它脏器中具有促红细胞生成以外的其它重要功能。EPO可能作为一种中枢神经系统炎症病理变化中的保护性细胞因子，对脑内的免疫反应有保护作用，这一作用可能通过影响脑内细胞因子的合成并发挥生物效应来实现。研究表明外周静脉或腹腔内注入重组人促红细胞生成素(recombinant human erythropoietin，rhEPO)能通过血脑屏障而直接在脑内产生神经保护作用。目前国内外对EPO与脑损伤的关系及机制研究主要集中于EPO对缺氧缺血性脑损伤的神经保护作用，而EPO与早产儿WMD的关系以及相关机制研究目前仍较少。
因此，本研究通过建立宫内感染导致WMD的新生大鼠模型，并给宫内感染后的新生大鼠腹腔注射一定剂量的rhEPO，观察rhEPO干预对宫内感染后的新生大鼠脑白质中的2′，3′-环核苷3′-磷酸二脂酶(2′，3′-cyclic nucleotide 3′phosphodiesterase，CNPase)、神经微丝(neurofilament，NF)和胶质纤维酸性蛋白(glial fibrillary acidicprotein，GFAP)表达水平的影响；观察rhEPO干预对宫内感染后的新生大鼠脑内细胞因子和诱导型一氧化氮合酶(inducible nitrie oxide synthase，iNOS)的基因表达以及NO含量的影响，从细胞、分子水平来说明EPO对宫内感染导致的新生大鼠WMD可能存在的治疗作用。该研究将为临床治疗早产儿WMD提供丰富的动物实验信息及依据，为今后进一步研究早产儿WMD的防治提供新的途径。
方法：
1．实验分组：对照组1(A组)、对照组2(B组)、宫内感染组(C组)、宫内感染+rhEPO低剂量治疗组(D组)、宫内感染+rhEPO中剂量治疗组(E组)、宫内感染+rhEPO高剂量治疗组(F组)，每组8只SD孕鼠。对照组1为正常对照，对照组2为胎龄15天的孕鼠左右两侧宫颈内各注射生理盐水0．2 mL，宫内感染组为胎龄15天的孕鼠左右两侧宫颈内各注射大肠杆菌稀释液0．2 mL，宫内感染+rhEPO低(中、高)剂量治疗组为宫内感染母鼠分娩后，新生大鼠于生后即刻腹腔注射1000(3000、5000)IU／kg rhEPO。
2．标本采集：按照新生1、3、7日龄分别随机选取各组中的新生大鼠经断头处死，迅速取出脑组织，将脑组织中性甲醛固定、石蜡包埋，或将脑组织经液氮速冻后-80℃冰箱中保存。
3．实验方法：应用苏木素-伊红(hematoxylin eosin，HE)染色观察各组新生1、3、7日龄大鼠脑室周围白质组织病理变化特点。免疫组化染色检测各组新生1、3、7日龄大鼠脑组织CNPase、NF和GFAP的表达，Western blot检测各组新生1、3、7日龄大鼠脑组织CNPase、NF和GFAP的蛋白水平，荧光定量RT-PCR检测各组新生1、3、7日龄大鼠脑内细胞因子肿瘤坏死因子(tumor necrosis factor，TNF)-α、白细胞介素(interieukin，IL)-1β、巨噬细胞炎性蛋白(macrophageinflammatoryprotein，MIP)-1α、MIP-1βmRNA和iNOSmRNA的表达，硝酸还原酶法检测各组新生1、3、7日龄大鼠脑内的NO含量。
结果：
1．48只孕鼠活动、进食均正常，无1例发生死亡。孕鼠分娩情况：A组共92只幼鼠，B组共85只幼鼠，C组共84只幼鼠，D组共89只幼鼠，E组共95只幼鼠，F组共78只幼鼠。宫内大肠杆菌感染的孕鼠子宫壁及胎盘内血管充血、水肿，并见大量中性粒细胞浸润。对照组的孕鼠子宫及病理检测均未见明显的炎症反应。
2．新生7日龄大鼠脑室周围白质部位HE染色可见：A、B两组脑白质组织结构正常，染色清晰；C、D两组脑白质染色淡，结构稀疏呈筛网状改变；E组脑白质染色偏淡，结构稀疏化程度较C组有所减轻；F组脑白质染色清晰，结构稀疏化程度较C组明显减轻。
3．宫内感染后的新生7日龄大鼠脑组织CNPase、NF表达显著下降，rhEPO治疗使宫内感染后的新生7日龄大鼠脑组织CNPase、NF表达有所上升。C组新生7日龄大鼠脑组织CNPase、NF蛋白水平较A、B两组均显著降低，E、F两组CNPase蛋白水平则较C组显著升高，F组NF蛋白水平亦较C组显著升高。宫内感染后的新生7日龄大鼠脑组织GFAP表达显著上升，rhEPO治疗使宫内感染后的新生7日龄大鼠脑组织GFAP表达有所下降。C组新生7日龄大鼠脑组织GFAP蛋白水平较A、B两组显著升高，F组GFAP蛋白水平则较C组显著降低。
4．宫内感染后新生1日龄大鼠脑内TNF-αmRNA的表达水平显著升高，中、高剂量的rhEPO治疗使宫内感染后新生1日龄大鼠脑内TNF-αmRNA的表达水平显著下降。宫内感染后新生1、3日龄大鼠脑内IL-1βmRNA的表达水平显著升高，中、高剂量的rhEPO治疗使宫内感染后新生大鼠1日龄脑内IL-1βmRNA的表达水平显著下降。宫内感染后新生1、3日龄大鼠脑内MIP-1αmRNA的表达水平显著升高，高剂量的rhEPO治疗使宫内感染后新生1、3日龄大鼠脑内MIP-1αmRNA的表达水平显著下降。宫内感染后新生1日龄大鼠脑内MIP-1βmRNA的表达水平显著升高，中、高剂量的rhEPO治疗使宫内感染后新生1日龄大鼠脑内MIP-1βmRNA的表达水平显著下降。
5．宫内感染后新生1、3日龄大鼠脑内iNOS mRNA的表达水平显著升高，中、高剂量的rhEPO治疗使宫内感染后新生1、3日龄大鼠脑内iNOS mRNA的表达水平显著下降。宫内感染后新生1、3日龄大鼠脑内NO含量显著升高，高剂量的rhEPO治疗使宫内感染后新生1、3日龄大鼠脑内NO含量显著下降。
结论：
1．宫颈接种大肠杆菌建立的官内感染大鼠模型能导致新生大鼠脑白质结构产生稀疏化表现，宫内感染后新生大鼠的脑白质组织存在少突胶质细胞损伤、轴突损害和反应性星形胶质化，提示宫内感染能够导致新生大鼠WMD。
2．通过给宫内大肠杆菌感染后的新生大鼠生后即刻腹腔注射一定剂量的rhEPO能够减轻宫内感染后新生大鼠脑白质出现的少突胶质细胞损伤、轴突损害和反应性星形胶质化，提示EPO对宫内感染导致的新生大鼠WMD具有一定的治疗作用。
3．宫内大肠杆菌感染后新生大鼠脑内的细胞因子TNF-α、IL-1β、MIP-1α和MIP-1βmRNA在生后早期有一过性表达增高的特点，提示由宫内感染导致的细胞因子级联反应在WMD的发生、发展中起着关键作用。
4．通过给宫内大肠杆菌感染后的新生大鼠生后即刻腹腔注射一定剂量的rhEPO可以抑制宫内感染后新生大鼠脑内细胞因子TNF-α、IL-1β、MIP-1α和MIP-1βmRNA的一过性表达增高，提示在宫内感染致新生大鼠WMD的早期炎症反应阶段，EPO可能通过抑制宫内感染后新生大鼠脑内细胞因子的大量表达，减轻炎症反应，从而起到保护作用。
5．宫内大肠杆菌感染后新生大鼠脑内iNOS mRNA在生后早期有一过性表达增高的特点，并相应出现NO含量的一过性升高，提示iNOS与其诱导产生的NO在宫内感染导致早产儿WMD的过程中可能起重要作用。
6．通过给宫内大肠杆菌感染后的新生大鼠生后即刻腹腔注射一定剂量的rhEPO可以抑制宫内感染后新生大鼠脑内iNOS mRNA的一过性表达增高和NO含量的一过性升高，提示在宫内感染致新生大鼠WMD的生后早期阶段，抑制iNOS的表达和NO的过量生成可能是EPO发挥治疗宫内感染导致的WMD的主要作用机制之一。
Objective:
White mater damage(WMD)in the preterm infant is the most important risk factorfor cerebral palsy,which brings heavy burdens to family and our society.Although theexact mechanism involved in WMD still remains unclear,the epidemiologic evidenceand study on animal models supported the view that one of the most important prenatalfactors that associated with WMD appeared to be intrauterine infection.The majorneuropathology of WMD consists of white mater astrocytosis,oligodendrocyte injury,myelination retardation and axonal damage.Recent studies showed a close relationshipbetween WMD and cytokines in the brain.Several mechanisms by which cytokines leadto WMD are recognized.Cytokines could(1)cause damage to oligodendrocyte,astrocyte,myelin,and axon;(2)stimulate other cytokines to cause further damage;(3)mediate the neurotoxicity of nitric oxide(NO).Therefore,the mechanism of intrauterineinfection caused WMD is supposed to be a series of changes occurred in cells andproteins induced by cytokines in the brain.
Recently,erythropoietin(EPO)has been shown to be beneficial in the brain and other organs.EPO might act as a protective cytokine in inflammatory pathologies of thecentral nervous system,and this protective effect may be carried out by influencing theproduction of cytokines after intrauterine infection.Studies have demonstratedrecombinant human erythropoietin(rhEPO)administered to the systemic circulationcould cross the blood-brain barrier and directly exert its neuroprotective effect in thebrain.Most of the recent studies focused on the effects of EPO on hypoxic-ischemicencephalopathy,and there were few reports on whether EPO had protective effectagainst WMD in the preterm infant.
In the present study,a rat model of intrauterine Escherichia coli infection wasestablished and a single intraperitoneal injection of rhEPO was given to the neonatalrats immediately after birth.In order to investigate the possible ameliorating effect ofrhEPO on WMD in developing rat brain after intrauterine infection,we examinedalterations in protein level of 2',3'-cyclic nucleotide 3'-phosphodiesterase(CNPase),neurofilament(NF)and glial fibrillary acidic protein(GFAP),and expression ofproinflammatory cytokines mRNA and inducible nitric oxide synthase(iNOS)mRNAas well as concentration of NO in the neonatal brain.This study may offer lots ofexperimental information and evidence on the clinical treatment of preterm WMD andmay provide new insight into the future therapeutic method.
Methods:
1.Group:control group 1(A),control group 2(B),intrauterine infection group(C),intrauterine infection plus low-dose rhEPO treated group(D),intrauterine infection plusmoderate-dose rhEPO treated group(E),intrauterine infection plus high-dose rhEPOtreated group(F).In intrauterine infection group,pregnant rats at 15 days of gestationwere inoculated endocervically with 0.4 mL of Escherichia coli suspension.Controlgroup 1 is the normal control group,and in control group 2,the rats were injected endocervically with 0.4 mL of sterile saline solution instead.In intrauterine infectionplus low(moderate,high)-dose rhEPO treated group,following matemal Escherichiacoli inoculation,the neonatal rats received a single intraperitoneal injection of rhEPO ata dose of 1000(3000,5000)IU/kg body weight immediately after birth.
2.Sample collection:The neonatal rats were sacrificed by decapitation at postnatalday 1(P1),P3 and P7,and the brains were fixed in neutral formaldehyde or stored at-80℃freezer.
3.Experimental methods:Hematoxylin-eosin(HE)staining determined thecerebral WMD of neonatal rats at P1,P3 and P7.Immunohistochemistry and westernblot analysis were used for evaluation of CNPase,NF and GFAP protein levels inneonatal rat brains at P1,P3 and P7.Real-time quantitative RT-PCR was used toanalyze necrosis factor(TNF)-α,interleukin(IL)-1β,macrophage inflammatory protein(MIP)-1αand MIP-1βmRNA as well as iNOS mRNA expression in neonatal rat brainsat P1,P3 and P7.Nitrate reductase method was used for detection of NO concentrationin neonatal rat brains at P1,P3 and P7.
Results
1.All the dams had normal food intake and activity,and none died afterinoculation.After delivery,92 live pups from group A,85 live pups from group B,84live pups from group C,89 live pups from group D,95 live pups from group E and 78live pups from group F were eligible for the study.Intrauterine Escherichia coliinoculation produced inflammation of uterus and placenta,but all cases of control grouphad no histologic evidence of intrauterine infection.
2.HE staining procedures revealed clear staining and normal structure ofperiventricular white matter from P7 rats of group A and B.Weak staining and focalrarefaction of periventricular white matter were shown in P7 rats of group C and D.HE staining of periventricular white matter from P7 rats in group E was still weak butslightly stronger than those in group C and D.The staining of periventricular whitematter from P7 rats in group F became obviously stronger than those in group C and D.
3.At P7,significant decreases in protein levels of CNPase and NF in neontal ratbrains of the intrauterine infection group were observed compared with the controls,however,rhEPO treatment markedly increased the protein levels of CNPase and NF inneontal rat brains as compared with the intrauterine infection group.GFAP protein levelwas markedly increased in neonatal rat brains of intrauterine infection group at P7 whencompared with the controls.In intrauterine infection plus high-dose rhEPO treatedgroup,the protein level of GFAP in neonatal rat brains at P7 was significantly decreasedas compared with the intrauterine infection group.
4.After intrauterine infection,the mRNA level of TNF-αin neonatal rat brains wassignificantly higher than the controls at P1.Moderate and high-dose rhEPO treatmentremarkably reduced intrauterine infection-induced elevation in TNF-αmRNA level atP1.In intrauterine infection group,the expression of IL-1βmRNA increased in neonatalrat brains at P1 and P3.Moderate and high-dose rhEPO treatment also significantlyreduced intrauterine infection-induced elevation in IL-1βmRNA level at P1.ThemRNA level of MIP-1αin intrauterine infection group increased at P1 and P3 ascompared with the controls,and MIP-1βmRNA was also significantly higher than thosein the controls at P1.High-dose rhEPO treatment could decrease the elevated expressionof MIP-1αmRNA after intrauterine infection at P1 and P3.Moderate and high-doserhEPO treatment also reduced the increased MIP-1βmRNA after intrauterine infectionat P1.
5.The expression of iNOS mRNA and concentration of NO in neonatal rat brainsof the intrauterine infection group were higher than those in the controls at P1 and P3. Moderate and high-dose rhEPO treatment could reduce intrauterine infection-inducedelevation in iNOS mRNA at P1 and P3,and high-dose rhEPO treatment could decreasethe elevated concentration of NO after intrauterine infection at P1 and P3.
Conclusion:
1.Focal rarefaction of cerebral white matter and changes in protein levels ofCNPase,NF and GFAP in neonatal rat brains after maternally endocervical Escherichiacoli inoculation,suggest that oligodendrocyte injury,axonal degeneration and reactiveastrogliosis really occurred in cerebral white matter and intrauterine infection could leadto WMD in neonatal rat brain.
2.Intraperitoneal injection ofrhEPO at a certain dose immediately after birth couldameliorate oligodendrocyte injury,axonal degeneration and reactive astrogliosis causedby intrauterine Escherichia coli infection,suggesting that EPO administration may exerta protective effect against WMD in neonatal rat brain after intrauterine infection.
3.The transient increase in mRNA levels of TNF-α,IL-1β,MIP-1αand MIP-1βinneonatal rat brains after intrauterine Escherichia coli infection,suggests that thecytokine cascade induced by intrauterine infection may play a pivotal role in thegeneration and development of WMD.
4.Intraperitoneal injection of rhEPO at a certain dose immediately after birth couldreduce intrauterine infection-induced elevation in mRNA levels of TNF-α,IL-1β,MIP-1αand MIP-1βin neonatal rat brains,suggesting that EPO administration mayexert a protective effect against WMD by inhibiting cytokine induction and attenuatinginflammation in the early stage after intrauterine infection.
5.The transient increase in iNOS mRNA level followed by the going-up NOconcentration after intrauterine Escherichia coli infection suggests that iNOS and NOmight play an important role in intrauterine infection induced WMD.
6.Intraperitoneal injection of rhEPO at a certain dose immediately after birth couldreduce intrauterine infection-induced elevations in iNOS mRNA level and NOconcentration,suggesting that inhibition of iNOS expression and NO overproduction inthe early stage of inflammation may be one of the mechanisms invovled in theprotective effect of EPO against WMD after intrauterine infection.
早产儿脑白质损伤(white matter damage，WMD)被认为是脑瘫发生的最主要的危险因素，给家庭和社会带来了巨大的负担。目前，早产儿WMD的机制尚不十分清楚，流行病学和动物实验研究均表明宫内感染在早产儿WMD的发生机制中起重要作用。WMD的病理特点主要是脑白质的凝固性坏死，坏死部位出现反应性星形胶质化、少突胶质细胞的丢失、髓鞘损伤和轴突损害。近年来研究认为，WMD的发生与脑内细胞因子的关系非常密切。细胞因子能以多种机制导致脑白质损伤：1．损伤少突胶质细胞、星形胶质细胞、髓鞘和轴突等；2．通过诱导其他细胞因子而进一步产生细胞损害；3．介导一氧化氮(nitric oxide，NO)的神经毒性作用等。官内感染导致早产儿WMD的机制可以推测为由细胞因子介导的一系列脑组织中各种细胞和蛋白合成的变化所致。
近年来的研究发现促红细胞生成素(erythropoietin，EPO)在脑和其它脏器中具有促红细胞生成以外的其它重要功能。EPO可能作为一种中枢神经系统炎症病理变化中的保护性细胞因子，对脑内的免疫反应有保护作用，这一作用可能通过影响脑内细胞因子的合成并发挥生物效应来实现。研究表明外周静脉或腹腔内注入重组人促红细胞生成素(recombinant human erythropoietin，rhEPO)能通过血脑屏障而直接在脑内产生神经保护作用。目前国内外对EPO与脑损伤的关系及机制研究主要集中于EPO对缺氧缺血性脑损伤的神经保护作用，而EPO与早产儿WMD的关系以及相关机制研究目前仍较少。
因此，本研究通过建立宫内感染导致WMD的新生大鼠模型，并给宫内感染后的新生大鼠腹腔注射一定剂量的rhEPO，观察rhEPO干预对宫内感染后的新生大鼠脑白质中的2′，3′-环核苷3′-磷酸二脂酶(2′，3′-cyclic nucleotide 3′phosphodiesterase，CNPase)、神经微丝(neurofilament，NF)和胶质纤维酸性蛋白(glial fibrillary acidicprotein，GFAP)表达水平的影响；观察rhEPO干预对宫内感染后的新生大鼠脑内细胞因子和诱导型一氧化氮合酶(inducible nitrie oxide synthase，iNOS)的基因表达以及NO含量的影响，从细胞、分子水平来说明EPO对宫内感染导致的新生大鼠WMD可能存在的治疗作用。该研究将为临床治疗早产儿WMD提供丰富的动物实验信息及依据，为今后进一步研究早产儿WMD的防治提供新的途径。
方法：
1．实验分组：对照组1(A组)、对照组2(B组)、宫内感染组(C组)、宫内感染+rhEPO低剂量治疗组(D组)、宫内感染+rhEPO中剂量治疗组(E组)、宫内感染+rhEPO高剂量治疗组(F组)，每组8只SD孕鼠。对照组1为正常对照，对照组2为胎龄15天的孕鼠左右两侧宫颈内各注射生理盐水0．2 mL，宫内感染组为胎龄15天的孕鼠左右两侧宫颈内各注射大肠杆菌稀释液0．2 mL，宫内感染+rhEPO低(中、高)剂量治疗组为宫内感染母鼠分娩后，新生大鼠于生后即刻腹腔注射1000(3000、5000)IU／kg rhEPO。
2．标本采集：按照新生1、3、7日龄分别随机选取各组中的新生大鼠经断头处死，迅速取出脑组织，将脑组织中性甲醛固定、石蜡包埋，或将脑组织经液氮速冻后-80℃冰箱中保存。
3．实验方法：应用苏木素-伊红(hematoxylin eosin，HE)染色观察各组新生1、3、7日龄大鼠脑室周围白质组织病理变化特点。免疫组化染色检测各组新生1、3、7日龄大鼠脑组织CNPase、NF和GFAP的表达，Western blot检测各组新生1、3、7日龄大鼠脑组织CNPase、NF和GFAP的蛋白水平，荧光定量RT-PCR检测各组新生1、3、7日龄大鼠脑内细胞因子肿瘤坏死因子(tumor necrosis factor，TNF)-α、白细胞介素(interieukin，IL)-1β、巨噬细胞炎性蛋白(macrophageinflammatoryprotein，MIP)-1α、MIP-1βmRNA和iNOSmRNA的表达，硝酸还原酶法检测各组新生1、3、7日龄大鼠脑内的NO含量。
结果：
1．48只孕鼠活动、进食均正常，无1例发生死亡。孕鼠分娩情况：A组共92只幼鼠，B组共85只幼鼠，C组共84只幼鼠，D组共89只幼鼠，E组共95只幼鼠，F组共78只幼鼠。宫内大肠杆菌感染的孕鼠子宫壁及胎盘内血管充血、水肿，并见大量中性粒细胞浸润。对照组的孕鼠子宫及病理检测均未见明显的炎症反应。
2．新生7日龄大鼠脑室周围白质部位HE染色可见：A、B两组脑白质组织结构正常，染色清晰；C、D两组脑白质染色淡，结构稀疏呈筛网状改变；E组脑白质染色偏淡，结构稀疏化程度较C组有所减轻；F组脑白质染色清晰，结构稀疏化程度较C组明显减轻。
3．宫内感染后的新生7日龄大鼠脑组织CNPase、NF表达显著下降，rhEPO治疗使宫内感染后的新生7日龄大鼠脑组织CNPase、NF表达有所上升。C组新生7日龄大鼠脑组织CNPase、NF蛋白水平较A、B两组均显著降低，E、F两组CNPase蛋白水平则较C组显著升高，F组NF蛋白水平亦较C组显著升高。宫内感染后的新生7日龄大鼠脑组织GFAP表达显著上升，rhEPO治疗使宫内感染后的新生7日龄大鼠脑组织GFAP表达有所下降。C组新生7日龄大鼠脑组织GFAP蛋白水平较A、B两组显著升高，F组GFAP蛋白水平则较C组显著降低。
4．宫内感染后新生1日龄大鼠脑内TNF-αmRNA的表达水平显著升高，中、高剂量的rhEPO治疗使宫内感染后新生1日龄大鼠脑内TNF-αmRNA的表达水平显著下降。宫内感染后新生1、3日龄大鼠脑内IL-1βmRNA的表达水平显著升高，中、高剂量的rhEPO治疗使宫内感染后新生大鼠1日龄脑内IL-1βmRNA的表达水平显著下降。宫内感染后新生1、3日龄大鼠脑内MIP-1αmRNA的表达水平显著升高，高剂量的rhEPO治疗使宫内感染后新生1、3日龄大鼠脑内MIP-1αmRNA的表达水平显著下降。宫内感染后新生1日龄大鼠脑内MIP-1βmRNA的表达水平显著升高，中、高剂量的rhEPO治疗使宫内感染后新生1日龄大鼠脑内MIP-1βmRNA的表达水平显著下降。
5．宫内感染后新生1、3日龄大鼠脑内iNOS mRNA的表达水平显著升高，中、高剂量的rhEPO治疗使宫内感染后新生1、3日龄大鼠脑内iNOS mRNA的表达水平显著下降。宫内感染后新生1、3日龄大鼠脑内NO含量显著升高，高剂量的rhEPO治疗使宫内感染后新生1、3日龄大鼠脑内NO含量显著下降。
结论：
1．宫颈接种大肠杆菌建立的官内感染大鼠模型能导致新生大鼠脑白质结构产生稀疏化表现，宫内感染后新生大鼠的脑白质组织存在少突胶质细胞损伤、轴突损害和反应性星形胶质化，提示宫内感染能够导致新生大鼠WMD。
2．通过给宫内大肠杆菌感染后的新生大鼠生后即刻腹腔注射一定剂量的rhEPO能够减轻宫内感染后新生大鼠脑白质出现的少突胶质细胞损伤、轴突损害和反应性星形胶质化，提示EPO对宫内感染导致的新生大鼠WMD具有一定的治疗作用。
3．宫内大肠杆菌感染后新生大鼠脑内的细胞因子TNF-α、IL-1β、MIP-1α和MIP-1βmRNA在生后早期有一过性表达增高的特点，提示由宫内感染导致的细胞因子级联反应在WMD的发生、发展中起着关键作用。
4．通过给宫内大肠杆菌感染后的新生大鼠生后即刻腹腔注射一定剂量的rhEPO可以抑制宫内感染后新生大鼠脑内细胞因子TNF-α、IL-1β、MIP-1α和MIP-1βmRNA的一过性表达增高，提示在宫内感染致新生大鼠WMD的早期炎症反应阶段，EPO可能通过抑制宫内感染后新生大鼠脑内细胞因子的大量表达，减轻炎症反应，从而起到保护作用。
5．宫内大肠杆菌感染后新生大鼠脑内iNOS mRNA在生后早期有一过性表达增高的特点，并相应出现NO含量的一过性升高，提示iNOS与其诱导产生的NO在宫内感染导致早产儿WMD的过程中可能起重要作用。
6．通过给宫内大肠杆菌感染后的新生大鼠生后即刻腹腔注射一定剂量的rhEPO可以抑制宫内感染后新生大鼠脑内iNOS mRNA的一过性表达增高和NO含量的一过性升高，提示在宫内感染致新生大鼠WMD的生后早期阶段，抑制iNOS的表达和NO的过量生成可能是EPO发挥治疗宫内感染导致的WMD的主要作用机制之一。
Objective:
White mater damage(WMD)in the preterm infant is the most important risk factorfor cerebral palsy,which brings heavy burdens to family and our society.Although theexact mechanism involved in WMD still remains unclear,the epidemiologic evidenceand study on animal models supported the view that one of the most important prenatalfactors that associated with WMD appeared to be intrauterine infection.The majorneuropathology of WMD consists of white mater astrocytosis,oligodendrocyte injury,myelination retardation and axonal damage.Recent studies showed a close relationshipbetween WMD and cytokines in the brain.Several mechanisms by which cytokines leadto WMD are recognized.Cytokines could(1)cause damage to oligodendrocyte,astrocyte,myelin,and axon;(2)stimulate other cytokines to cause further damage;(3)mediate the neurotoxicity of nitric oxide(NO).Therefore,the mechanism of intrauterineinfection caused WMD is supposed to be a series of changes occurred in cells andproteins induced by cytokines in the brain.
Recently,erythropoietin(EPO)has been shown to be beneficial in the brain and other organs.EPO might act as a protective cytokine in inflammatory pathologies of thecentral nervous system,and this protective effect may be carried out by influencing theproduction of cytokines after intrauterine infection.Studies have demonstratedrecombinant human erythropoietin(rhEPO)administered to the systemic circulationcould cross the blood-brain barrier and directly exert its neuroprotective effect in thebrain.Most of the recent studies focused on the effects of EPO on hypoxic-ischemicencephalopathy,and there were few reports on whether EPO had protective effectagainst WMD in the preterm infant.
In the present study,a rat model of intrauterine Escherichia coli infection wasestablished and a single intraperitoneal injection of rhEPO was given to the neonatalrats immediately after birth.In order to investigate the possible ameliorating effect ofrhEPO on WMD in developing rat brain after intrauterine infection,we examinedalterations in protein level of 2',3'-cyclic nucleotide 3'-phosphodiesterase(CNPase),neurofilament(NF)and glial fibrillary acidic protein(GFAP),and expression ofproinflammatory cytokines mRNA and inducible nitric oxide synthase(iNOS)mRNAas well as concentration of NO in the neonatal brain.This study may offer lots ofexperimental information and evidence on the clinical treatment of preterm WMD andmay provide new insight into the future therapeutic method.
Methods:
1.Group:control group 1(A),control group 2(B),intrauterine infection group(C),intrauterine infection plus low-dose rhEPO treated group(D),intrauterine infection plusmoderate-dose rhEPO treated group(E),intrauterine infection plus high-dose rhEPOtreated group(F).In intrauterine infection group,pregnant rats at 15 days of gestationwere inoculated endocervically with 0.4 mL of Escherichia coli suspension.Controlgroup 1 is the normal control group,and in control group 2,the rats were injected endocervically with 0.4 mL of sterile saline solution instead.In intrauterine infectionplus low(moderate,high)-dose rhEPO treated group,following matemal Escherichiacoli inoculation,the neonatal rats received a single intraperitoneal injection of rhEPO ata dose of 1000(3000,5000)IU/kg body weight immediately after birth.
2.Sample collection:The neonatal rats were sacrificed by decapitation at postnatalday 1(P1),P3 and P7,and the brains were fixed in neutral formaldehyde or stored at-80℃freezer.
3.Experimental methods:Hematoxylin-eosin(HE)staining determined thecerebral WMD of neonatal rats at P1,P3 and P7.Immunohistochemistry and westernblot analysis were used for evaluation of CNPase,NF and GFAP protein levels inneonatal rat brains at P1,P3 and P7.Real-time quantitative RT-PCR was used toanalyze necrosis factor(TNF)-α,interleukin(IL)-1β,macrophage inflammatory protein(MIP)-1αand MIP-1βmRNA as well as iNOS mRNA expression in neonatal rat brainsat P1,P3 and P7.Nitrate reductase method was used for detection of NO concentrationin neonatal rat brains at P1,P3 and P7.
Results
1.All the dams had normal food intake and activity,and none died afterinoculation.After delivery,92 live pups from group A,85 live pups from group B,84live pups from group C,89 live pups from group D,95 live pups from group E and 78live pups from group F were eligible for the study.Intrauterine Escherichia coliinoculation produced inflammation of uterus and placenta,but all cases of control grouphad no histologic evidence of intrauterine infection.
2.HE staining procedures revealed clear staining and normal structure ofperiventricular white matter from P7 rats of group A and B.Weak staining and focalrarefaction of periventricular white matter were shown in P7 rats of group C and D.HE staining of periventricular white matter from P7 rats in group E was still weak butslightly stronger than those in group C and D.The staining of periventricular whitematter from P7 rats in group F became obviously stronger than those in group C and D.
3.At P7,significant decreases in protein levels of CNPase and NF in neontal ratbrains of the intrauterine infection group were observed compared with the controls,however,rhEPO treatment markedly increased the protein levels of CNPase and NF inneontal rat brains as compared with the intrauterine infection group.GFAP protein levelwas markedly increased in neonatal rat brains of intrauterine infection group at P7 whencompared with the controls.In intrauterine infection plus high-dose rhEPO treatedgroup,the protein level of GFAP in neonatal rat brains at P7 was significantly decreasedas compared with the intrauterine infection group.
4.After intrauterine infection,the mRNA level of TNF-αin neonatal rat brains wassignificantly higher than the controls at P1.Moderate and high-dose rhEPO treatmentremarkably reduced intrauterine infection-induced elevation in TNF-αmRNA level atP1.In intrauterine infection group,the expression of IL-1βmRNA increased in neonatalrat brains at P1 and P3.Moderate and high-dose rhEPO treatment also significantlyreduced intrauterine infection-induced elevation in IL-1βmRNA level at P1.ThemRNA level of MIP-1αin intrauterine infection group increased at P1 and P3 ascompared with the controls,and MIP-1βmRNA was also significantly higher than thosein the controls at P1.High-dose rhEPO treatment could decrease the elevated expressionof MIP-1αmRNA after intrauterine infection at P1 and P3.Moderate and high-doserhEPO treatment also reduced the increased MIP-1βmRNA after intrauterine infectionat P1.
5.The expression of iNOS mRNA and concentration of NO in neonatal rat brainsof the intrauterine infection group were higher than those in the controls at P1 and P3. Moderate and high-dose rhEPO treatment could reduce intrauterine infection-inducedelevation in iNOS mRNA at P1 and P3,and high-dose rhEPO treatment could decreasethe elevated concentration of NO after intrauterine infection at P1 and P3.
Conclusion:
1.Focal rarefaction of cerebral white matter and changes in protein levels ofCNPase,NF and GFAP in neonatal rat brains after maternally endocervical Escherichiacoli inoculation,suggest that oligodendrocyte injury,axonal degeneration and reactiveastrogliosis really occurred in cerebral white matter and intrauterine infection could leadto WMD in neonatal rat brain.
2.Intraperitoneal injection ofrhEPO at a certain dose immediately after birth couldameliorate oligodendrocyte injury,axonal degeneration and reactive astrogliosis causedby intrauterine Escherichia coli infection,suggesting that EPO administration may exerta protective effect against WMD in neonatal rat brain after intrauterine infection.
3.The transient increase in mRNA levels of TNF-α,IL-1β,MIP-1αand MIP-1βinneonatal rat brains after intrauterine Escherichia coli infection,suggests that thecytokine cascade induced by intrauterine infection may play a pivotal role in thegeneration and development of WMD.
4.Intraperitoneal injection of rhEPO at a certain dose immediately after birth couldreduce intrauterine infection-induced elevation in mRNA levels of TNF-α,IL-1β,MIP-1αand MIP-1βin neonatal rat brains,suggesting that EPO administration mayexert a protective effect against WMD by inhibiting cytokine induction and attenuatinginflammation in the early stage after intrauterine infection.
5.The transient increase in iNOS mRNA level followed by the going-up NOconcentration after intrauterine Escherichia coli infection suggests that iNOS and NOmight play an important role in intrauterine infection induced WMD.
6.Intraperitoneal injection of rhEPO at a certain dose immediately after birth couldreduce intrauterine infection-induced elevations in iNOS mRNA level and NOconcentration,suggesting that inhibition of iNOS expression and NO overproduction inthe early stage of inflammation may be one of the mechanisms invovled in theprotective effect of EPO against WMD after intrauterine infection.
引文
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[2] Rezaie P, Dean A. Periventricular leukomalacia, inflammation and white matter lesions within the developing nervous system[J]. Neuropathology, 2002, 22(3):106-132.
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