Mr Imaging Evaluate AQP4 Gene Silence To Treat Early Hypoxic Ischemic Brain Damage In Vivo

Neonatal hypoxic-ischemic encephalopathy(HIE) is a main cause of acute neonatal death and children’s mental retardation, cerebral palsy, epilepsy and other nervous system injury. Brain edema is the early pathological changes of the death of brain cell, thus the effective prevention and treatment has important clinical significance. Aquaporin 4(AQP4) plays an important role in the central osmotic pressure adjusting and brain water balance.Therefore, AQP4 is a decisive factor in the development of brain edema.With the rapid development of gene therapy for nearly a decade,AQP4 gene knockout technology can be realized in neonatal rats.And this technique,which provides a new way for the treatment of cerebral edema, can reduce the symptoms of water intoxication and brain edema caused by hypoxia ischemia.However, the evaluation method of gene therapy is still unsatisfactory. How to evaluate assessment of gene expression non-invasively is the key problem which must be faced with gene therapy.Currently, Magnetic resonance diffusion imaging( DWI) is the only method to measure and image water molecule diffusion in vivo. DWI can also provide quantifiable apparent diffusion coefficient(ADC), which opens up a new way for the study of the pathological mechanism of brain edema and gene therapy effect assessment.In recent years, RNA interference(RNAi),as a specific gene produces blocking Biotechnology,is able to down specific gene expression quickly and efficiently.Thus, RNAi has become a research tool for molecular biology which could replace knockout. In order to further study the mechanism in brain edema formation of AQP4 and assess the treatment effect of AQP4 gene silence on hypoxic-ischemic brain edema.We intend to use 3~5 days old York pigs to establish a new born pig model of ischemic hypoxic. Using RNA interference technology, we will import the si RNA into animals’ body through route of cerebrospinal fluid. After that, we use brain MRI scanner to observe changes in brain at different times after hypoxia ischemia. And then to seek new clues of the pathogenesis and treatment of cerebral edema on HIE.Objective1. To observe the early changes of DWI images and ADC values of newborn piglets with acute hypoxic ischemic brain damage( HIBD).2. To investigate the correlation between ADC value and AQP4 expression in the early process of HIBD in neonatal pigs3. To evaluate the therapeutic effect of AQP4 gene silence HIBD of new pigs.Methods1. The piglets were randomly divided into experimental group 10, control group 12. The experimental group was given HIBD model preparation, the control group only received sham operation. Respectively before hypoxia ischemia and after hypoxia ischemia in 1 h, 3 h, 6 h, 9 h and 12 h for routine MR scanning and DWI examination and get TIWI, T2 WI, T2-flair, DWI images and corresponding ADC image.Observe the change of images and select subcortical and cortical, periventricular area and hippocampus areas for ADC measurement. In the experimental group, at different time points after ischemia and hypoxia 2 piglets were sacrificed once the MR check is completed. The piglets were done nerve behavioral score before sacrifice,and for pathological examination of brain after.2. The piglets were randomly divided into experimental group 24, control group 24. The experimental group was given HIBD model preparation, the control group only received sham operation. Respectively before hypoxia ischemia and after hypoxia ischemia in 1 h, 3 h, 6 h, 9 h and 12 h for routine MR scanning and get DWI images and corresponding ADC image. Observe the change of images and select subcortical and cortical, periventricular area and hippocampus areas for ADC measurement. Four piglets in the experimental group and contrast each time after ischemia anoxic after MR examination were executed.Take the brain do immunohistochemical analysis, detection of AQP4 protein expression. Then, take the brain tissue do immunohistochemical analysis to detect the AQP4 protein expression.3. The piglets were randomly divided into 4 groups: 11 pigs in group A( the experimental group), 11 pigs in group B(the control group1), 6 pigs in group C(the control group2), and 4 pigs in group D(the control group 3). Group A were given si RNA-AQP4 by cerebrospinal fluid injection, ischemia and hypoxia. Group B were given si RNA-Negative(AQP4 non interference sequence) by cerebrospinal fluid injection, ischemia and hypoxia. Group C were given si RNA-Negative((AQP4 non interference sequence) by cerebrospinal fluid injection and sham-operation. Group D were given no treatment. Every group were taken to have MR scanning before and after ischemia and hypoxia in 1h, 3h, 6h, 9h and 12 h to get DWI and ADC graph. The changes of DWI were observed and then three areas were chosen to have the measurement of ADC value and take the score of neurological which are cortex and subcortical, periventricular area and the hippocampus. Each animal’s brain in these groups were taken after 12 h, then taken to have the Real Time PCR exam and immunohistochemistry which could measure the expression of AQP4 protein and AQP4 m RNA. The arterial blood was extracted to have the blood gas analysis and exam the enzymes of the brain.Results1.The scores of neurological behavior of the experimental group after ischemia and hypoxia were significantly reduced than the control group(P<0.05). The preparation of HIBD model of new born pigs was successful. The experimental group and the control group both showed no unusual signal on T1 WI, T2 WI and T2-flair.The experimental group showed punctiform high signal areas on DWI after 1 hour of ischemia and hypoxia, with the extension of time the range and the intensity of the high signal areas was obviously expanded on the graph of DWI after ischemia and hypoxia for 3h, 6h and 9h, these high signal areas mainly were distributed in cortex and subcortical, periventricular area and the hippocampus. After 12 hours of ischemia and hypoxia it showed intracranial diffuse high signal changes on DWI. The ADC value of each part of the experimental group was obviously reduced after 1 hour and reached to the minimum after 3 hours, and it rose again after 6h, 9h and 12 h but it remained lower than the level of ADC value before ischemia and hypoxia. The control group showed no changes in ADC value on DWI at different time. After ischemia and hypoxia at 1h, 3h, 6h, 9h and 12 h, the ADC value of the experimental group was reduced than the control group and the differences was significant(P<0.05). On pathology, part of the intracellular edema happened after ischemia and hypoxia at 1 hour, and its range and intensity aggravated after 3 hours, then the vasogenic edema happened after 6 hours. At 12 hours after ischemia and hypoxia the vasogenic edema aggravated, but the intracellular edema remained mainly.2.The immunohistochemistry showed the expression quantity of AQP4 protein was increased than the control group after ischemia and hypoxia at 1h, 3h, 6h, 9h and 12 h on parts of the experimental group which were periventricular area and the hippocampus and the difference was significant(P<0.05). The expression quantity of AQP4 protein at each time point was negatively correlated with the ADC value of the corresponding time(P<0.05).3. Group A, C and D showed no obvious unusual high signal on DWI at each time point and group B showed the same results to the former two researches on DWI. The ADC value of each part of group A showed slightly reduced after ischemia and hypoxia at each time point, and almost recovered at 12 h, and it was higher than group B, the difference was significant(P<0.05). The neurological behavior score showed that group A had higher score than group B at each time point after ischemia and hypoxia and the difference was significant(P<0.05). The immunohistochemistry showed that group A had fewer amount of AQP4 protein staning cells than group B. The result of Real Time PCR showed that the relative expression amount of AQP4 m RNA was obviously reduced in group A than group B in the area of the hippocampus, and the difference was significant(P<0.05). The relative expression amount of AQP4 m RNA was obviously reduced in group A than group B in the area of periventricular, but the difference was not significant(P=0.25); The relative expression amount of AQP4 m RNA in group A and B of the hippocampus was more than that of the perientricular area and the difference was significant(P<0.05).Conclusions1. DWI is sensitive and effective in examination methods of new-born piglets at early stage of HIBD, and the changes of ADC value could show the pathophysiological changes better than DWI images.2. The AQP4 expression at early HIBD of new-born piglets shows high and is negative correlation with ADC value.3. RNAi could inhibit the expression of AQP4 gene effectively, it could relieve the symptoms of HIBD and could be an effective method of gene therapy.4. The expression of AQP4 in the hippocampus is more than that in the periventricular area, and it could be a basic reason why the hippocampus is more sensitive to ischemia and hypoxia than other areas.5. The measurement of ADC value could be an effective method of dynamic evaluation in vivo for the treatment of HIBD by AQP4 gene silence.