Protective Effect Of Erythropoietin On White Matter Damage In Neonatal Rat Brain After Intrauterine Infection

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.Results1.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.