| Objective: Germinal matrix-intraventricular hemorrhage(GM-IVH)is one of the most common complications of premature infants.Due to the fragile germinal matrix vasculature of premature children,coupled with hemodynamics and respiratory instability,it is often prone to spontaneous bleeding.Bleeding hematoma oppression of brain tissue white matter can lead to intracranial hypertension,ion overload,increased oxygen free radicals and other changes,producing a series of pathophysiological changes simultaneously,such as inflammation,demyelination,neuronal apoptosis,causing irreversible injury on ventricular white matter,which in turn leads to a poor prognosis on children,and brings heavy economic and psychological burden.The study found that in human premature infants,white matter lesions display an abundance of hyaluronan(HA),arrested maturation of oligodendrocyte precursor cells(OPCs),and reduced myelination.Similarly,animal demyelination experiments found that HA can inhibit the maturation of OPCs and myelination.It is currently known that GM-IVH can induce the perivascular white matter to produce inflammation and HA aggregation.We hypothesized that HA may also be involved in the development of OPCs after GM-IVH hinder the formation of white matter myelination.Hyaluronidase(Hyaluronidase,HAase)can be decomposed HA,GM-IVH of premature infants,brain injection application HAase degradation of HA,whether to reduce the obstacle of the development of brain tissue OPCs the HA and relieve patients with cerebral white matter myelination obstacles? There have been few reports on this subject at home and abroad.This experiment is based on the animal model of premature rabbit GM-IVH,and HAase was injected into the brain of the model animal,and the control group was set up.There are two observed indicators:(1)Neuron-glial antigen2(NG2),which can reflect the maturation of OPCs;(2)Myelin basic protein(MBP):MBP is recognized changes in CNS reflect the presence or absence of more specific biochemical indicators of myelinosis.The purpose of this experiment is to observe whether HAase treatment can improve the obstruction of OPC in premature rabbit brain in GM-IVH and reduce the myelin degeneration in periventricular white matter,so as to achieve the neuroprotective effect and provide a new idea for the clinical treatment of premature infant GM-IVH.Methods:1.Making GM-IVH premature rabbit model: the normal pregnancy of New Zealand rabbits is 32 days,selectd with pregnancy for 29 days New Zealand white rabbits,The born rabbits are premature rabbits.80 New Zealand white rabbits were used,only weight 30-55 g.Offering heat preservation,milk powder immediately after the birth.Premature rabbits were randomly divided into control group,model group(pure GM-IVH model),the treatment group(GM-IVH model + HAase treatment)three groups.Within 3 hours after birth,the model group and the treatment group received 50% glycerolintraperitoneal injection with a dose of 10ml/Kg to induce the occurrence of GM-IVH.The normal group does not give any treatment.All animal experiment operating process are in accordance with the international animal ethical norms set by the executive committee.2.Establishment and treatment of haemorrhage:(1)all preterm preterm rabbits were subjected to a cephalic ultrasound 24 hours after birth to verify the occurrence of intracranial hemorrhage.(2)The rabbits in the treatment group were given intracerebroventricular injection of HAase under the guidance of ultrasound.On the 2nd,4th and 6th day after birth,the rabbits in the treatment group received HAase injected into the lateral forebrain by a dose of 20 ul / kg.(3)model group according to the above method to the lateral ventricle injection of the same dose of saline,normal group without any treatment.3.Extraction of brain tissue: Three groups of preterm rabbits in normal group,model group and HAase treatment group were randomly sacrificed on the 3rd,7th and 14 th days after birth,The rabbits were sacrificed and the brain tissues were rapidly mobilized.The obtained samples were stored in a-80 refrigerator for Western blotting.The remaining specimens were fixed ?in 4% paraformaldehyde for immunohistochemical staining and hematoxylin-eosin staining.4.Immunohistochemical method was adopted to observe whether there was difference between the expression of NG2 in the samples of the three groups.5.Westrn blot was adopted to compare the expression the MBP in the samples of the three groups.6.Statistical method: SPSS19.0 statistical analysis software and Microsoft Excel 2003 were used for statistics processing.The measurement data were expressed as mean ± standard deviation(LSD),LSD test was used for comparison among groups.If they do not meet the homogeneity of variance,use rank sum.The test standard was 0.05,when P<0.05,it had statistical significance.Results:1.Successful copy of GM-IVH animal modelResults of brain ultrasound examination: 30 patients in the model group,5 were not bleeding,4 died during the experiment,and 21 were actually involved in the experiment;There were 29 patients in the treatment group,3 were not bleeding,and 5 deaths during the experiment,21 were actually involved in the experiment.The normal group of 21 had no bleeding,and no death occurred during the experiment,and 21 were actually involved in the experiment.HE staining results: GM-IVH success of the IVH preterm birth: when rabbit brain tissue by optical microscope ventricle visible around a large number of red blood cells and inflammatory cells infiltration,bleeding not group no significant change,confirmed the rabbit model of GM-IVH premature preparation of success.2.The expression of NG2 in the brain tissue of the premature rabbits.This study found that NG2 cells expression is visible in each new rabbit around ventricular premature brain white matter regions.The expression of NG2 cell quantity reduce gradually in the same set of comparison over time,In the normal group,the difference was statistically significant at 3 days and at 7 days,at 14 days after birth(P <0.05),and there was no statistically compared at 7 days with 14 days after birth(P>0.05).The HAase treatment group and model group in 3 days,7 days,14 days after birth,pairwise comparison,group differences had statistically significance(P <0.05);At the same time point comparison,the expression level of NG2 cells is model group> normal group> treated group in 3 days and 14 days after birth,at 7 days after birth,the expression level of NG2 cells is Model group> treatment group> normal group.No matter at 3 days,7 days or 14 days after birth,any two of the three groups of any time comparison,the differences were statistically significant.3 The expression of MBP in the brain tissue of the premature rabbits.In this study,the expression of MBP protein was observed in the white matter areas around the ventricle.The MBP protein expression in three groups of premature rabbits increased with the increase of age,and the variance analysis was statistically significant(P < 0.05).The expression of MBP in normal group and HAase group was significantly different at 3 days,7 days and 14 days after birth(P <0.05).At 14 days,the expression of MBP level of the treatment group was higher than the normal group,the difference was statistically significant(P <0.05).The expression of MBP in brain tissue of model group was increased at 3 days,at 7 days and 14 days after birth,and there was no significant compared 3 day with 7 day(P > 0.05);The expression of MBP at 14 days was higher than 3 days and 7 days,and the difference was significantly(P <0.05).At the same time-point,the expression of MBP is HAase treatment group > model group.Analysis of variance show that there was not significant difference on expression level of MBP among the three groups.Conclusion:The expression of NG2 and MBP in brain tissue decreased after GM-IVH in premature rabbits,indicating that GM-IVH hinder oligodendrocyte precursor cell development and myelination;When intracerebroventricular injection of hyaluronidase was carried out on GM-IVH premature rabbits,it was found that the expression activity of NG2 and MBP in the Treatment Group was higher than that in the Model Group,indicating that hyaluronidase was beneficial to the development of oligodendrocytes and the formation of myelin. |
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