| Objective: Germinal matrix-intraventricular hemorrhage(GM-IVH)is the most common intracranial hemorrhage in the neonatal period,and also the main cause of white matter damage in premature infants.With the progress of medical science,the survival rate of super premature infants is gradually increasing,while the number of children with GM-IVH is also going up.The children with serious GM-IVH would suffer cerebral palsy,learning disabilities,cognitive disorder and other neurological sequelae.Even mild intracranial hemorrhage would also have a certain impact on the prognosis of child patients,and currently,there is no effective treatment.In recent years,with the further research on the secondary injury of intracranial hemorrhage,we found that the secondary inflammatory response of the post intraventricular hemorrhage would lead to irreversible damage to brain tissue.Hyaluronic acid is the main component of extracellular matrix,and also an inflammatory medium which would induce inflammatory response when it is bound with the receptor CD44.Hyaluronic acid accumulation could be found in many inflammatory diseases.Hyaluronidase is specific hydrolases of hyaluronic acid.Research has proved that hyaluronidase could be used in the treatment of inflammatory diseases of the central nervous system.However,there are also little report at home or abroad about whether the hyaluronidase could reduce the secondary inflammatory injury of the post intraventricular hemorrhage.This experiment,by the animal model of premature rabbit GM-IVH,aims to discuss: 1.the expression of hyaluronic acid receptor CD44 and TNF-a in the brain tissue of premature rabbit GM-IVH;2.the expression of hyaluronic acid receptor CD44 and TNF-a in the brain tissue of premature rabbit GM-IVH after the intervention of hyaluronic acid.Methods:1 42 premature rabbits were selected with pregnancy period of 29 days(normal pregnancy period is 32 days)given birth by New Zealand white rabbit,warmed them and fed them with milk.Then randomly divided into three groups: Normal Group,Model Group(GM-IVH model only)and Treatment Group(GM-IVH model+hyaluronidase),and each group had 14 premature rabbits.The steps of intraperitoneal injection of glycerin to inducing GM-IVH,prepare premature rabbit GM-IVH model were taken as reference.Specific steps were as follows: intraperitoneal injection of 50% glycerin(dose of10ml/Kg)to premature rabbits in Model Group and Treatment Group within 3hours after birth,GM-IVH model was made,but nothing was done to the Normal Group.The cranial ultrasonic examination was carried out for the premature rabbits of the three groups within 1 days after birth to check whether there was intracranial hemorrhage in the Treatment Group and Model Group.The lateral cerebral ventricle injection of hyaluronidase(1U/u L)with dose of 20 u L/kg was required for the premature rabbits in the Treatment Group on the second,fourth and sixth day;while the lateral cerebral ventricle injection of normal saline with dose of 20?L/kg was required for the premature rabbits in the Model Group on the same day,and nothing was done to the Normal Group.The premature rabbits were taken care in each groups.2 Premature rabbits in each group were randomly divided into two sub-groups,with 7 premature rabbits respectively for each subgroup.On the3 rd and 7th day,the premature rabbits in each subgroup were sacrificed by anesthesia and their brain tissues were peeled rapidly;after observation of the sample state,periventricular brain tissues were extracted rapidly and the samples were marked;some samples were put into refrigerator with 80 degrees below zero for westrn blot.Remaining samples were immersed in 4%paraformaldehyde and fixed for later immunohistochemical staining and HE staining.3 Immunohistochemical method was adopted to observe whether there was difference between the expression of TNF-a and that of CD44 in the samples of the three groups.Westrn blot was adopted to compare the expression of TNF-a with that of CD44.4 Statistical method: SPSS17.0 statistical analysis software and Microsoft Excel 2003 were used for statistics processing.If the measurement data was normal destribution,then presented by mean ± standard deviation(x ±SD).Homogeneity test of variance was carried out,if the homogeneity of variance was satisfied,Independent Samples Test was adopted for the comparison of the two sample mean,LSD for the comparison of multiple sample mean;otherwise,rank-sum test was adopted.The test standard was 0.05,when P<0.05,it had statistical significance.Results:1 Successful copy of GM-IVH animal modelCesarean was performed on pregnant rabbit on its 29 th day to obtain premature rabbits.Intraperitoneal injection of glycerol was given within 3hours after being born.Craniocerebral ultrasound,pathologic samples and microscope were used to prove that the premature rabbit GM-IVH animal model was copied successfully.2 The expression of CD44 in the brain tissue of the premature rabbits.The expression of CD44 in the Model Group reached the highest level on the 3rd day but reduced on the 7th day,and the difference had statistical significance(P<0.05).The expression of CD44 in the Treatment Group and Normal Group was higher on the 7th day compared to that on the 3rd day,and the difference had statistical significance(P<0.05).On the same day,the expression of CD44 in Model Group was highest,while lowest in Normal Group and the Treatment Group lied between,and the difference had statistical significance(P< 0.05).3 The expression of TNF-a in the brain tissue of the premature rabbits.The expression of TNF-a in the Model Group and Treatment Group was higher on the 3rd day compared to that on the 7th day,and the difference had statistical significance(P<0.05).The expression of TNF-a in the Normal Group on the 3rd day and on the 7th day had no difference(P<0.05).On thesame day(3rd day or 7th day),the expression of TNF-a in Model Group was highest,while lowest in Normal Group and the Treatment Group lied between,and the difference had statistical significance(P< 0.05).Conclusion:If the premature rabbits suffered GM-IVH,TNF-a content in brain tissue would increase,indicating that GM-IVH had secondary inflammatory response and the response would reduce as time went by.In addition,the premature rabbits post GM-IVH showed high expression of hyaluronic acid receptor CD44 and the expression would reduce as time went by,indicating that there was hyaluronic acid accumulation in the post GM-IVH brain tissue,and the hyaluronic acid could participate in the secondary inflammatory response in post GM-IVH after binding with its receptor CD44.When intracerebroventricular injection of hyaluronidase was carried out on GM-IVH premature rabbits,it was found that the expression activity of TNF-a in the Treatment Group was lower than that in the Model Group,indicating that hyaluronidase could reduce the secondary inflammatory response in post GM-IVH;while the expression of CD44 in the Treatment Group was also decreased compared with the Model Group,indicating that hyaluronidase could inhibit the expression activity of CD44 through hydrolyzed hyaluronic acid so as to alleviate the secondary inflammatory response in post GM-IVH. |
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