The Mechanism Study Of Axon Development Disorders And Synaptic Deficit Mediated By IL-1? In Septic Neonatal Rats

Neonatal sepsis has seriously threatened the survival and health of children for ages,which could cause long-term neurological dysfunction.During perinatal period,the brain is still on developmental stage with incomplete blood brain barrier,thus serious infection is able to spread to central nervous system,leading to neuronal inflammation,and subsequent axonal and synaptic lesions.This might be one of the reasons for neuronal dysfunction induced by neonatal sepsis,however the specific molecular mechanism is still unclear.Microglia,the main inflammatory cell in the brain,could be activated by severe infection and stress,generating excess amounts of pro-inflammatory cytokines,including interleukin-1?,which is one of the major pro-infammatory cytokines.It has been documented that IL-1? is involved in all kinds of CNS pathogenesis.P38-MAPK,which is a signaling pathway closely associated with inflammation,could be activated by IL-1?.Therefore it is hypothesized that microglia-derived IL-1? could induce axon development disorders and synaptic deficit through p38-MAPK during neonatal sepsis.Object:To investigate whether lipopolysaccharide(LPS)administration to neonatal rat would result in axon development disorders and synaptic deficit,and to explore whether LPS administration could activate microglia to overexpress IL-1? in vivo.To prove IL-1? could downregulate the expression of essential proteins related to axon and synaptic via p38-MAPK in vitro.Method:1.One day old SD rats were randomly divided into two groups.The rats in experimental group were intraperitoneally injected with LPS,while the rats in control group were intraperitoneally injected with PBS.Proteins including PLP,NFL,NFM,NFH,Synaptophysin,PSD95 associated with axon,synapse and myelin sheath were detected by immunofluorescence and immunoblotting at 7 days,14 days and 28 days after LPS injection.The microstructure change was observed by EM at 28 days after LPS injection.Imunofluorescence and Nissl staining were used to detect apoptosis of cortical neurons.2.One day old SD rats were randomly divided into two groups.The rats in experimental group were intraperitoneally injected with LPS,while the rats in control group were intraperitoneally injected with PBS.Activated microglia was detected by immunofluorescence.IL-1? and IL-1R1 in periventricular white matter(PWM)were detected by immunofluorescence and immunoblotting methods at 6 hours,2 days,4 days and 6 days after LPS injection.3.Primary cultured neurons were administrated with interleukin-1? in an ideal concentration.Western blot and immunofluorescence were used to determine the expressions of proteins related to axon(NFL,NFM)and synapse(Synaptophysin).4.Interleukin-1? was administrated to intervene primary cultured neurons,and then we studied the activation of p38-MAPK signal pathway by Western blot.Using the inhibitor SB203580 of p38-MAPK signal pathway,primary cultured neurons were divided into four groups respectively:control group,interleukin-1? group,interleukin-1? +SB203580 group and SB203580 group.The expressions of proteins related to axon and synapse were detected by Western blot to confirm that p38-MAPK signaling pathway mediated the axon and synapse deficit induced by interleukin-1?.Results:1.PLP,NFL,NFM,NFH and Synaptophysin protein expression was markedly decreased in the CC at 7,14,and 28 days after LPS administration when compared with the matching controls(P<0.05).By electron microscopy,at 28 days after LPS injection,the packing density of myelinated axons was decreased significantly in the CC when compared with the corresponding controls under both low and high magnification.Ultrastructural study also showed damaged myelin sheath and Ranvier node at 28 days after LPS injection when compared with the intact myelin sheath and Ranvier node in the corresponding control.Additionally,in LPS-injected rats,the diameter of axons was noticeably smaller at 28 days in comparison with that of the matching controls(P<0.05).Moreover,swollen synaptic vesicles,damaged endoplasmic reticulum and mitochondrial cristae were observed in some of the neurons and axon in the cortex at 28 days after LPS injection.There was no significant difference in Nissl's staining and apoptotic neurons in the cerebral cortex of the frontal lobe at 7,14,and 28 days after LPS injection compared with corresponding controls.2.After intraperitoneal injection of LPS for 6 hours,2 days,4 days and 6 days,the microglia in corpus callosum were activated with a round or oval shape and increased number.Microglia-derived interleukin-1? was significantly increased in the LPS group compared with control(P<0.05).At the same time,the corresponding receptor IL-1R1 also up-regulated in the neuronal axon in the LPS group compared with control(P<0.05).3.The expressions of axon and synapse related protein(NFL,NFM,Synaptophysin)in primary cultured neurons were markedly decreased after interleukin-1?administration comparing with the control group(P<0.05),while IL-1R1 antagonist could antagonize the down-regulation of interleukin-1?(P<0.05).4.Interleukin-1? administration could significantly up-regulate the phosphorylation of p38-MAPK in 0.5 hour in primary cultured neurons(P<0.05),and the inhibitor of p38-MAPK signal pathway,SB203580,could significantly reverse the down-regulation of axon and synapse related protein of neurons(NFL,NFM,Synaptophysin)(P<0.05).Conclusion:Microglia-derived interleukin-1? could inhibit the expression of neuronal axon and synapse related proteins through activating p38-MAPK,and it might give a partial view of the mechanism of neuronal axon and synapse injury caused by neonatal sepsis.