The Expression Of TLR4 And TNF-α During Lipopolysaccharide-induced White Matter Damage In The Early Neonatal Rat And The Protection Of Betamethasone

ObjectiveWhite matter damage (WMD) is an important form of brain injury in very low birth weight infants, often with the high incidence and bad prognosis. Increasing evidence indicates that intrauterine infection and the responses to inflammation may be the major contributors to WMD. It have been proved that WMD developes during the high time of myelination and oligodendrocytes progenitors(preOL) are indentified as a putative target cell. Neonatal Wistar rats inflammatory models of postnatal day 2( P2) were produced by LPS injected in-traperitoneally in this research to explore the change of apoptosis cell number in the white matter, the influence on mylination and the expression of TLR4 and TNF-α in the brain tissue of neonatal rats by morphological and molecular biological methods, and to investigate whether betamethasone could protect brain from being injuried.MethodsThe neonatal rats were separated into groups randomly, group A in which different doses of LPS ( group A₁, 1.0mg/kg,group A₂,2.5 mg/kg,group A₃, 5.0mg/kg) were injected intraperitoneally on P2; group B was betamethasone treated group in which LPS 5.0mg/kg and different doses of betamethasone (2.0mg/kg or 4. 0mg/kg) were injected intraperitoneally at the same time(B₁, 2.0mg/kg) or 16h before LPS injection on P2(B₂, 2.0mg/kg; B₃, 4.0mg/kg) ; group C in which LPS 5.Omg/kg was injected on P7; group D was control group in which equal amount of normal saline was injected intraperitoneally on P2. Apoptosis cells in neonatal rats brain of every group were measured after the injection 24h by Tunel. The expression of CNPase at 24h after injection and MBP at P14 in brain tissue of every group were measured by immunohistochem-istry. The expression of TLR4 and TNF-a in fetal brain tissue of group A3 was measured at lh, 2h, 3h, 4h, 6h and express of TLR4 at 2h and TNF-ctat 4h after the injection in different dose of LPS was measured by RT-PCR and Immuno-histochemistry.Results(l)The mortatlity was 12. 5% (one out of eight) after LPS treatment at 24h after injection in group A3. 28. 6% (two out of seven) of the survival rats can be seen punctate hemorrhage in the corpus callosum(CC) , external capsule and/or intraventricular hemorrhage. Hemmorrhage can be seen in the brain of group Bl in the area of exernal capsule or even in pallium, white matter under the pallium and candate putamen. On P14, inteneration can be seen in CC and increased glial cells were observed around the inteneration area in group A3, but not in other groups. (2)Apoptosis cells which were stained to brown color were observed in CC after injection for 24h. There was no change in group A, ( P > 0.05 ). Following LPS treatment at the increasing dosage, there were the more counts of apoptosis cells than those in group D. It has been shown significantly different between group A2, A3 and group D(6.38 ±0.74vs4.88 ± 1.25, P <0. 05; 21.43 ± 1.72vs4. 88 ± 1.25, P <0.01). Compared with those of group A3, counts of apoptosis cells in group B, were increased(23.13 ± 1.13 vs 21.43 ± 1.72, P < 0.05), while those in the Beta pretreatment group were significantly decreased and there were fewer counts of apoptosis cells with the increasing dosage of Beta (13.63 ±1.06 vs21.43±1.72,P<0.01;5.63±1.06 vs 21.43 ±1.72,P< 0.01). Compared with those of group A3, counts of apoptosis cells in group C were significantly decreased(8.75 ± 1. 28vs21.43 ± 1.72, P < 0.01). (E>There was an acute increasing loss of CNPase positive cells following the increasingdosage of LPS treatment at 24h after injection. (^Following LPS treatment to neonatal rats, the protein expression of MBP in neonatal brain decreased evidently compared with the corresponding control group at P14. (§)TLR4 positive cells were found in the lpetomeninges, choroids plexus and corpus callosum after LPS treatment. There was a down regulation of the expression of TLR4 protein and TLR4mRNA following LPS treatment in group A3. At lh, it has been shown lower than that of group D ( P < 0.05). It was at 2h when the nadir value was reached (P<0.01). At3h, it was increasing gradually although it was still significantly lower than that of group D ( P <0.01) , until 4h there was no different with that of group D. Following LPS treatment (at the increasing dosage of 1.0 mg/kg, 2.5 mg/kg and 5. Omg/kg) , the TLR4 protein and TLR4mRNA level were not changed in group A, ( P >0. 05 ) , while they were shown significantly decreased at 2h after LPS treatement in group A2 and A3 with the increasing dosage of LPS compared with those of group D (P < 0. 01). Compared with group A3, the expression of brain TLR4 did not changed or decreased in group B; at 2h after LPS treatement, while they were shown significantly increased in group B2 and B3 with the increasing dosage of Beta compared with those of group A3, the expression of TLR4 in group C were significantly increased. ?The expression of TNF-a protein and TNF-amRNA increased following LPS treatment in group A3. At4h, the peak was reached, afterwards it gradually decreased. Following LPS treatment at increasing dose at 4h after injection, TNF-a expression was low in group A!, there was no different with group D( P >0. 05) , while it was significantly higher than that of group D following the dose increasing (P < 0. 01). Compared with that of group A3, the expression of brain TNF-a increased in group B, at 4h after LPS treatement( P <0.05 ) , while they were shown significantly decreased in group B2 and B3 with the increasing dosage of Beta compared with those of group' A3, the expression of TNF-a in group C were significantly decreased(P<0.01).ConclusionsIt have suggested that LPS-induced inflammation could affect the develop-ment of myelination in neonatal brain, and contribute to WMD. TLR4 were perhaps mediated the WMD in the neonatal rats induced by LPS, TNF-ct could be the key cytokine which mediated the injury, and they may reflect the degree of the damage. Pretreatment with betamethasone could decrease the counts of apop-tosis cells and hinder the damage to myelination. Our data also verified that at P7 the white matter could display greater resistance to injury than that of P2. The possibility is raised that pretreatment with betamethasone may blocking the innate immune response by targeting the TLR4 pathway and decreased the expression of TNF-a. These finding provide the basis for clinical approaches targeted at protecting the premature brain from inflammatory damage, which may prove beneficial for treating WMD.