| BackgroundCerebral ischemia is a major health crisis throughout the world, and the currently available thrombolytic therapy is unsatisfactory. Cell death following cerebral ischemia is mediated by a complex pathophysiological interaction of various mechanisms. During an ischemic insult, not only neurons but all of the components of the neurovascular unit, such as glia, endothelia, pericytes and basal membranes, are destroyed. Previous studies have shown that excessive stimulation of poly (ADP-ribose) polymerase (PARP-1) is crucial for cerebral injury after ischemic insult, which is an important cause of cell death in all cell types within the neurovascular unit.ObjectiveTo investigate the effects of Lentivirus-mediated PARP-1 siRNA transfer on neurovascular unit (NVU) in rats following cerebral ischemia.Methods1. Cell culture and transfectionNeuron cultures were prepared from the hippocampi of 24-hour-old Sprague Dawley rats and were infected the LV-scrambled-shRNA and LV-PARP1-shRNAs. The neurons were divided into five groups:the control group (CON, normal neurons), the negative control group (NC, LV-control-shRNA), and the siRNAl, siRNA2 and siRNA3 groups. PARP-1 expressions were analyzed using real-time PCR to select the siRNA knock-out PARP-1 mRNA for further animal experiments.2. Animal models152 rats were randomly divided into the following four groups:sham, Middle Cerebral Artery Occlusion (MCAO), MCAO treated with LV-control-shRNA (Control shRNA), and MCAO treated with LV-PARP1-shRNA (PARP-1 shRNA). The animals in each group were then assigned to 6 subgroups.Furthermore, an additional 18 rats were used to evaluate the suppression efficacy of LV-PARP1-shRNA after intracerebroventricular (i.c.v.) injection into the brain; the PARP-1 activity in another 18 rats was analyzed by western blotting.3. Neurological assessment was used to evaluate neurological deficits.4. TTC straining was conducted to assess infarction volume.5. Evans blue leakage and brain water content was used to evaluate blood brain barrier deficits.6. H&E straining was used to examine pathological changes of NVU.7. Electron microscopy was used to observe ultrastructure of NVU.Results1. SiRNA1, siRNA2 and siRNA3 all suppressed PARP-1 expression notably (p<0.01).2. PARP-1 enzymatic activity was dramatically increased at 1 h and 3 h post-MCAO compared with those in sham animals (p<0.01, p<0.01).3. Compared to control siRNA group, PARP-1 mRNA of LV-PARP1-siRNA rats decreased significantly (p<0.05).4. Compared to sham group, neurobehavioral scores of MCAO and Control siRNA increased significantly (p<0.05); Compared to MCAO group, neurobehavioral scores of LV-PARP1-siRNA rats decreased significantly (p<0.05).5. Compared to MCAO and Control siRNA group, infarction volume of LV-PARP1-siRNArats decreased significantly (9.875%±0.611%, p<0.05).6. Compared to sham group, EB leakage and brain water content of MCAO and Control siRNA increased significantly (p<0.05); A notable reduction of EB leakage and brain water content was observed in LV-PARP1-siRNA group compared to MCAO rats.7. Neurons of the MCAO were damaged and swollen with deeply stained nucleus, while neurons in the LV-PARP1-siRNA group were well-distributed and stained evenly.8. The scanning electron microscope results revealed that mitochondria was swollen and mitochondrial cristae was fractured, myelin became thinner and delaminated, endothelial cell and basal lamina were deformed, synapse was reduced and destroyed in the MCAO group. Ultrastructure deficits of NVU were ameliorated in LV-PARP1-siRNA group.ConclusionLentivirus-mediated PARP-1 siRNA transfer may ameliorate neurological function by alleviating NVU deficits and reducing infarction volume following cerebral ischemia. |
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