Effect Of Germinal Matrix-intraventricular Hemorrhage On Brain Tissue Glucose Transporters In Premature Rabbits

Objective:The germinal matrix-intraventricular hemorrhage is the most common types of intracranial hemorrhage in the premature infants. With the improvement of medical technology and nursing, the survival of preterm infants has increased dramatically over the past few decades, but to the low gestational age and low birth weight premature infants, the incidence of GM-IVH is still a major problem. GM-IVH have severe neurological disorders, such as epilepsy, cognitive abnormalities and cerebral palsy, so the GM-IVH and it’s neurological disorder is still a major public health problem in the worldwide. The mechanism of brain injury secondary to GM-IVH is still unclear.The GM-IVH in preterm infants often combined with lateral ventricle expansion, there is no effective methods to treatment the severe GM-IVH besides symptomatic treatment, lumbar puncture is one of the main methods of symptomatic treatment, the purpose is to release the bleeding, higher protein and excessive cerebrospinal fluid in the lateral intra-ventricle in order to keep the unimpeded of cerebrospinal fluid circulation and prevent the lateral ventricle continuing to expand, form serious hydrocephalus and reduce the oppression to the brain. There is a prominent phenomenon of the cerebrospinal fluid continuous detection by the methods of lumbar puncture, the glucose concentration in the cerebrospinal fluid is lower than the normal reference in preterm infants, but the blood glucose concentration is normal, Volpe summarized as follows: the first day after the hemorrhage,the glucose concentration in the cerebrospinal fluid was lower, the lowest appeared at 5-15 days after IVH, and always last for several weeks, even continued to three months later,which could not be explained by inflammation.The glucose is the main energy for the brain, but glucose from the blood into the brain cells must be mediated by GLUT1 and GLUT3. The classic patient with a low glucose concentration in the cerebrospinal fluid is glucose transporter type 1 deficiency syndrome(GLUTI-DS), the patient’s blood glucose concentration was normal, but the cerebrospinal fluid glucose to blood glucose ratio was low, brain CT and MRI also were normal, EEG showed slow wave background, more diffuse or focal abnormal discharge, it often resulted in infantile drug-resistant seizures, a complex movement disorder, progressive intellectual disability.The mechanism of lower cerebrospinal fluid glucose in GM-IVH babies is not clear and weather it involve the secondary brain injury after GM-IVH is also unclear? Initially it was thought to be due to the consumption of red blood cells and the increasing of the glycolysis, but after removal the red blood cells and the lactic concentration back normal in the cerebrospinal fluid, the glucose concentration continued at a low level, so it was rejected. some scholars doubt it may be due to the decreasing of glucose transport from blood to brain because of IVH.There is rare literature to report it.Using Western Blot, we detected the expression of GLUT1, GLUT3 protein in brain tissue in GM-IVH animal model, hope to explore the possible mechanism of reducing glucose concentration in cerebrospinal fluid after GM-IVH, and hope to provide some new therapeutic for GM-IVH babies.Methods:1 Premature rabbits were born by cesarean section, quickly took immature rabbit into the incubator, gave milk powder feeding after born,immature rabbit were randomly divided into two groups, Three hours later, the premature rabbits in one group were given intraperitoneally injection of 50% glycerol(10ml/kg),the other group did not receive any treatment.2 Two groups of premature rabbit respectively accepted cranial untrasoundexamination after birth 24 hours and observed the GM-IVH. 15 pups were recognized having GM-IVH which injected glycerol as Model group, 15 pups were recognized no GM-IVH which not injected glycerol as Control group. Within each group, 15 pups rabbits were randomly divided into three sub-groups: every time period had 5 pups(at 24 h, 48 h and 72 h after born). All the preterm rabbits were given anesthesia and then a part of the fresh brain tissue into 4% poly fixed Formaldehyde Solution, for later hematoxylin-eosin staining, to observe the morphological changes of brain cells after server GM-IVH, the other part of the fresh brain tissue immediately with liquid nitrogen frozen, then the specimen in-80 ℃ refrigerator, for Western blotting to detect the expression of GLUT1 protein and GLUT3 protein in control group and model group.3 Statistical analysis SPSS13.0 statistics software and Microsoft Excel 2003 were used to carry on statistics processing. Measurement data was performed with mean±standard deviation. If the data are homogeneity of variance,the comparison at different times were performed with LSD. Two independent sample mean test use Independent sample t-test. If they do not meet the homogeneity of variance, use rank sum. P-value<0.05(double side) has statistical significance.Results:1 Successfully established a premature rabbit model of severe GM-IVH.The head ultrasound screening, specimen, HE staining showed the intra-ventricular hemorrhage in preterm rabbits.2 The expression of GLUT1 in the brainThrough the western blot test, the expression of GLUT1 protein in the brain tissue around the ventricle in the control group were gradually reduced after born at 24 hour, 48 hour and 72 hour, all had difference(P<0.05). In model group, the expression of GLUT1 protein at 24 hour, 48 hour, 72 hout also had difference(P<0.05), the protein expression was the highest at 24 hour, followed by 48 hour, the last is at 72 hour after birth. In the model group, GLUT1 protein expression was lower than the same time point of the control group at 72 hour, but were higher than the same time point of the control group at 24 hour and 48 hour,all had significance(P<0.05).3 The expression of GLUT3 in the brainThrough the western blot test, the expression of GLUT3 protein in the brain tissue around the ventricle in the control group was gradually increased after broth at 24 hour, 48 hour and 72 hour, all had difference(P<0.05). In the model group, the expression of GLUT3 protein at 24 hour, 48 hour, 72 hour also had difference(P<0.05), the protein expression was the highest at 48 hour, followed by 24 hour, the last was 72 hour after birth. In the model group, GLUT3 protein expression was no significance compared with the same time point of the control group(P>0.05),but at 72 hour it was lower than the same time point of the control group, 48 hours was higher than the same time point of the control group, all had significance(P<0.05).Conclusions:1 Within 72 hour after birth,Along with the growth of age,the expression of GLUT1 protein in brain tissue was gradually reduced, and the expression of GLUT3 protein was gradually increased in the normal premature rabbit.2 Post-intraventricular hemorrhage, the expression of GLUT1 protein in brain had a temporary increasing representing a compensatory response, then it decreased possibly resulting in reducing the glucose concentration in cerebrospinal fluid.3 Post-intraventricular hemorrhage, the expression of GLUT3 protein in brain had a temporary increasing representing a protective reaction to IVH, then it decreased, suggested it could be involved in the secondary brain injury after severe GM-IVH.