Establishment Of Lipopolysaccharide-induced White Matter Injury Model In Rat Based On The "multi-hit" Theory And Research On Related Mechanisms

Objective White matter injury（WMI）is a common type of injury in premature infants and young children.It is a global health problem and the most common cause of cerebral palsy and cognitive deficits.In this study,the WMI model of Sprague-Dawley（SD）rats was established by lipopolysaccharide（LPS）repeated stimulation in the early stage of development,and the effects of multi-hit mediated by inflammatory response on histological and behavioral effects and related molecular mechanisms were investigated.MethodsAccording to the random number table method,32 SD rats were divided into the following 4 groups:（1）control group（n=8）;（2）LPS1（n=8）;（3）LPS2 group（n=8）;（4）LPS3 group（n=8）.Pregnant rats in the control group and LPS1 group were injected with 0.9% saline on the 18 th day of gestation,while the pregnant rats in the LPS2 and LPS3 groups were injected with 0.05mg/kg LPS and 0.1mg/kg LPS on the18 th day of gestation.The offspring rats of the control group were injected with 0.9%normal saline at the age of 20 days,and the offspring rats of the LPS1-3 groups were injected with 1 mg/kg LPS at the age of 20 days.Through repeated stimulation of LPS in the early stage of brain development,a model of white matter injury in SD rats induced by "multi-hit" mediated by inflammatory response was established,and the changes of related indicators and changes in related indicators were preliminarily explored through behavioral,histological,and molecular experiments in offspring rats.Possible mechanism of action of Notch signaling pathway.ResultsCompared with the control group,（1）LPS1-3 groups showed in the behavioral experiment that the forelimb suspension time was significantly reduced,the number of foot steps in the grid test was increased,and the latency time and total number of the Morris water maze test were increased.The distance increased significantly（P<0.05）.（2）hematoxylin-eosin（HE）staining observation: tissue structure disorder,cell pyknosis,and local lymphocyte infiltration.（3）Histochemical staining showed that the specific staining of Notch1,Jagged1,ionized calcium binding adaptor molecule 1（IBA1）and glial fibrillary acidic protein（GFAP）was significantly increased.（4）Under the transmission electron microscope（TEM）,the cells were observed to have different degrees of edema,the mitochondrial cristae were broken,and part of the nuclear membrane was damaged.（5）The results of molecular experiments indicated that the relative expressions of Notch1,Jagged1,IBA1,GFAP mRNA and protein were significantly increased,and the difference was statistically significant（P<0.05）.（6）The enzyme linked immunosorbent assay（ELISA）results showed that the expression levels of inflammatory factors interleukin-1β（IL-1β）and tumor necrosis factor-α（TNF-α）in the serum of LPS1-3 group mice were significant increased（P<0.05）.ConclusionIn this study,the risk factors of clinical white matter injury in early brain development were simulated,and the "multi-hit" white matter injury model in SD rats induced by LPS repeated stimulation was established.The findings were as follows: the pups in the LPS groups,there were not only pathological damage of brain tissue,but also developmental problems such as motor dysfunction and learning and memory deficits.The activation of glial cells caused by repeated inflammatory stimuli may be related to the upregulation of Notch1/Jagged1 pathway.