Of western blotting and cavity measurement was performed using one-way factorial analysis of variance (ANOVA) followed by Tukey’s post hoc test. Comparisons of the BBB scores of the three groups of rats were performed using two-way repeated ANOVA with Tukey’s post hoc test. A value of P,0.05 was regarded as statistically significant.Results Characteristics of PMWs used for Gene TransfectionTo evaluate the propagation characteristics of PMWs through the spinal cord, temporal pressure profiles of PMWs before andEffects of siRNA Sequences on Expression of GFAP and VimentinFigure 3 shows the expression levels of GFAP and vimentin in the two groups: (1) SCI alone (SCI group) and (2) scrambledTreatment of SCI by PMW-Mediated siRNA DeliveryFigure 2. Delivery of fluorescence-labeled siRNA into injured spinal cords. (A) Experimental arrangement for 17460038 PMW-based siRNA delivery into rat injured spinal cord. After laminectomy exposing a tenth dorsal vertebra followed by making a spinal contusion, the solution of siRNA was intrathecally injected around the lesion, to which PMWs were applied. (B) Distributions of fluorescence-labeled siRNA and GFAP expression in sagittal sections of injured spinal cords at five days after trauma for the three groups: SCI alone, SCI plus siRNA injection and SCI plus siRNA injection followed by application of 10 pulses of PMW generated at a laser fluence of 0.3 J/cm2. The enlarged and merged images show evident incorporation of siRNA into GFAP-positive astrocytes located in the anterior funiculus in the PMW application group (arrowheads). Scale bars represent 200 mm. (C) Depth dependence of fluorescence intensity from the fluorescence-labeled siRNA in injured spinal cords. Values are expressed as means 6 S.E.M (n = 9, each group). The total number of pixels showing green fluorescence in the SCI alone group indicates the level of background autofluorescence. (D) Depth dependence of the number of pixels showing yellow, which indicates colocalization of siRNA with GFAP-positive astrocytes in injured spinal cords. Values are expressed as means 6 S.E.M (n = 5, each group). doi:10.1371/journal.pone.0051744.gsiRNA injection after SCI (scrambled siRNA group). The expression levels of both IF proteins were similar in the two groups, indicating that the gene silencing effects in the following results were caused by the siRNA sequences.Delivery of siRNAs Targeting GFAP and Vimentin into Injured Spinal Cords using PMWsOn the basis of the results of the reporter siRNA delivery experiment described above, a mixture of siRNAs targeting GFAP and vimentin was delivered into the injured spinal cords of rats by applying PMWs. To examine the effects of transfection with these siRNAs, immunostaining for GFAP and vimentin was performed on sagittal sections of the spinal tissues at five days after SCI under three different conditions: (1) SCI group, (2) siRNA group, and (3) PMW group. In the SCI group, marked expressions of GFAP and vimentin were observed around the spinal contusion. Hesperidin site Comparatively, the most silencing of GFAP was observed in the PMW group (Fig. 4A). Immunostaining for vimentin revealed that the expression of vimentin in the injured spinal tissue of the 64849-39-4 custom synthesis PMWgroup was obviously suppressed (Fig. 4B). To investigate this effect more quantitatively, immunoblot analyses were performed using spinal cords treated under the three conditions described above and intact spinal tissues after laminectomy as a sham-treatment control (Fig. 4C, 4.Of western blotting and cavity measurement was performed using one-way factorial analysis of variance (ANOVA) followed by Tukey’s post hoc test. Comparisons of the BBB scores of the three groups of rats were performed using two-way repeated ANOVA with Tukey’s post hoc test. A value of P,0.05 was regarded as statistically significant.Results Characteristics of PMWs used for Gene TransfectionTo evaluate the propagation characteristics of PMWs through the spinal cord, temporal pressure profiles of PMWs before andEffects of siRNA Sequences on Expression of GFAP and VimentinFigure 3 shows the expression levels of GFAP and vimentin in the two groups: (1) SCI alone (SCI group) and (2) scrambledTreatment of SCI by PMW-Mediated siRNA DeliveryFigure 2. Delivery of fluorescence-labeled siRNA into injured spinal cords. (A) Experimental arrangement for 17460038 PMW-based siRNA delivery into rat injured spinal cord. After laminectomy exposing a tenth dorsal vertebra followed by making a spinal contusion, the solution of siRNA was intrathecally injected around the lesion, to which PMWs were applied. (B) Distributions of fluorescence-labeled siRNA and GFAP expression in sagittal sections of injured spinal cords at five days after trauma for the three groups: SCI alone, SCI plus siRNA injection and SCI plus siRNA injection followed by application of 10 pulses of PMW generated at a laser fluence of 0.3 J/cm2. The enlarged and merged images show evident incorporation of siRNA into GFAP-positive astrocytes located in the anterior funiculus in the PMW application group (arrowheads). Scale bars represent 200 mm. (C) Depth dependence of fluorescence intensity from the fluorescence-labeled siRNA in injured spinal cords. Values are expressed as means 6 S.E.M (n = 9, each group). The total number of pixels showing green fluorescence in the SCI alone group indicates the level of background autofluorescence. (D) Depth dependence of the number of pixels showing yellow, which indicates colocalization of siRNA with GFAP-positive astrocytes in injured spinal cords. Values are expressed as means 6 S.E.M (n = 5, each group). doi:10.1371/journal.pone.0051744.gsiRNA injection after SCI (scrambled siRNA group). The expression levels of both IF proteins were similar in the two groups, indicating that the gene silencing effects in the following results were caused by the siRNA sequences.Delivery of siRNAs Targeting GFAP and Vimentin into Injured Spinal Cords using PMWsOn the basis of the results of the reporter siRNA delivery experiment described above, a mixture of siRNAs targeting GFAP and vimentin was delivered into the injured spinal cords of rats by applying PMWs. To examine the effects of transfection with these siRNAs, immunostaining for GFAP and vimentin was performed on sagittal sections of the spinal tissues at five days after SCI under three different conditions: (1) SCI group, (2) siRNA group, and (3) PMW group. In the SCI group, marked expressions of GFAP and vimentin were observed around the spinal contusion. Comparatively, the most silencing of GFAP was observed in the PMW group (Fig. 4A). Immunostaining for vimentin revealed that the expression of vimentin in the injured spinal tissue of the PMWgroup was obviously suppressed (Fig. 4B). To investigate this effect more quantitatively, immunoblot analyses were performed using spinal cords treated under the three conditions described above and intact spinal tissues after laminectomy as a sham-treatment control (Fig. 4C, 4.
