Experimental Study Of GAD Gene Silence By Smeall Interfering RNA Injected Into Internus Segment Of Glubus Pallidus Of Parkinson’s Disease Rat

ObjectiveSmall interfering RNA (siRNA) was injected into the external segment ofglobus pallidus by using stereotaxic technology. To observe the changes inneuronal activities of the globus pallidus internus (GPi) and the improvementsof motor behavior and neurotransmitter contents of parkinson’s disease(PD)rat brain were detected after the injection of the siRNA-rGAD65gene. Weexplored a new injection target for Parkinson’s disease treatment.MethodsPD rats models were randomly divided into gene therapy group (receivedsiRNA-rGAB65), vector group (recerived AAV2blank vector) and blankcontrol groups(received PBS). There are24rats for each group. Behavioralanalysis, including rotation experiment, cylinder experiment and gaitexperiment, were conducted at4W,8W, and16W after injection in eachgroups. neuronal activities and firing rates of extracellular single unitary of theGPi were recorded.The transmitter contents of gamma-aminobutyricacid(GABA) and glutamic acid were detected by high performance liquidchromatography. The expression of GAD65were detected by western blot andimmunohistochemistry methods. TH positive neurons expression wereexamined by immunohistochemistry methods at4W,8W,16W after injectionrespectively. ResultsAt8W after6-OHDA damaged, the PD rat models had the behavior ofaverage speed>7rpm or rotating laps>210cycles per30min after apomorphineinjection. The gait experiment was used to observe the footprint of PD ratmodels. Compared with normal front limb, the activities of affected limb werereduced and the footprints were significant smaller than that of normal limbs.From4weeks to16weeks, rotating laps of gene therapy group weresignificantly decreased than that of the two control groups (P<0.001), but therewere no statistical significance difference between the two control groups(P>0.05). The firing pattern of the GPi neurons changed towards moreirregular in the normal rats,and less regular and bursty. The firing pattern of theGPi neurons changed towards more regular and bursty in the siRNA grouprats,and less irregular.Cylinder experiment was used to observe the utilizingrate of two forelimbs. The utilizing rate of right forelimb was only30%of leftforelimb in PD rat models. The utilization rate of limb was increased to40%,45%,55%at4weeks,8weeks and16weeks, respectively after treatment.There were more GAD65positive cells on the left side of rAAV2-rGAD65grouprats, but the rGAD65positive neurons were few on the left side of other twogroups rats and right side of all three groups. There were a trend that thenumber of GAD65postitive neurons were decreased from4weeks to16weeks after ioperation. The glutamic acid contents of GPi, detected by highperformance liquid chromatographic analysis, also had a decreasing trendafter injection from4weeks to16weeks, and significantly lower than that oftwo control groups. But the GABA content of STN was significant higher thanthat of two control groups from4weeks to16weeks. The GAD65expressionwas higher than that of the two control groups, but there had no statisticaldifference as compared the GAD65group with AAV2vector group or blankcontrol group (P>0.05). Conclusions1. SiRNA injection in GPi could improve rotating behavior, which inducedby apomorphine, and enhance the utilization rate of limb in PD rat models.2. SiRNA injection in GPi could decrease glutamate content of GPi andincrease the GABA content of STN.3. GPi inject siRNA GAD, silence, learn from the function on the certificate,can alleviate the symptoms of PD...