Peak pressure (20?51 MPa) and impulse (1.8?.3 Pa?s) were considered to be high enough for PMW-mediated gene transfection [33,35]. Because a siRNA solution was intrathecally injected to prevent damage to the 1485-00-3 spinal cord parenchyma, the diffusion and delivery of siRNAs into the parenchyma should be limited without PMW application. The results of fluorescence-labeled siRNA delivery showed that the depth of intense fluorescence from the siRNA was much increased by the application of PMWs; after subtracting the background autofluorescence, the integrated number of pixels showing green fluorescence in the spinal tissue following the application of PMWs was approximately 3.5 times larger than that with siRNA injection alone, achieving a depth of 1200?500 mm (Fig. 2C). The colocalization of siRNA with GFAP-positive astrocytes indicated that glial cells were efficiently transfected by the fluorescence-labeled siRNA in the anterior funiculus at five days after SCI (Fig. 2B, 2D). Moreover, the results of immunostaining showed that the most evident reduction of GFAP and vimentin was achieved around the spinal contusion in the PMW group at five days after SCI (Fig. 4A, 4B, 4C, 4D). These observations are attributable to the capability of PMWs to deliver siRNAs into deeply located astrocytes in the spinal contusion. As described above, astrocytes are activated after SCI with enlarged somas and intensive expression of IF proteins over time, and then a cystic cavity is formed in the region surrounded by the glial scar [21]. This neurodegenerative PZ-51 nature leads to a progressive increase in the size of the cavitation area [76,77]. The results of immunohistological analysis at five days post-SCI showed that GFAP and vimentin were markedly up-regulated around the lesion in the SCI group, resulting in prominent formation of glial scars and cavities at three weeks after injury (Fig. 5A, 5B, 6A). For effective reduction of glial scar formation, itis necessary to deliver the relevant siRNAs into a broad region in the spinal contusion, and then to reduce astrogliosis. At five days post-SCI, the IF proteins were silenced especially in the white matter of the anterior horns in the PMW group. This shows that PMWs enhanced the uptake of siRNA into glial cells, especially those located in the ventral funiculus. The anterograde tracing experiment showed numerous regenerating CST axons in the vicinity of the lesion in the PMW group (Fig. 7). This demonstrates that inhibition of excessive glial activity in the injured spinal tissue causes promotion of spontaneous axonal outgrowth, leading to functional recovery. The motor function of the lower limbs of rats in the PMW group was significantly improved from five days after injury (Fig. 8). These findings are consistent with the results of a previous study in which double mutant mice lacking GFAP and vimentin showed significant axonal regrowth of descending fibers of the corticospinal tract and ventral horn serotonergic tract, leading to an improved functional recovery after spinal cord hemisection [20]. To apply PMW-based siRNA therapy to larger animals with a thicker spinal column, deeper propagation of PMWs and broader distribution of therapeutic siRNAs are required. In our recent study, significant gene expression was observed in rat skin as a test tissue by applying PMWs that had to first propagate through 15-mm-thick tissue 12926553 phantoms, demonstrating the capability of PMWs for cell permeabilization in tissues of ,15 mm.Peak pressure (20?51 MPa) and impulse (1.8?.3 Pa?s) were considered to be high enough for PMW-mediated gene transfection [33,35]. Because a siRNA solution was intrathecally injected to prevent damage to the spinal cord parenchyma, the diffusion and delivery of siRNAs into the parenchyma should be limited without PMW application. The results of fluorescence-labeled siRNA delivery showed that the depth of intense fluorescence from the siRNA was much increased by the application of PMWs; after subtracting the background autofluorescence, the integrated number of pixels showing green fluorescence in the spinal tissue following the application of PMWs was approximately 3.5 times larger than that with siRNA injection alone, achieving a depth of 1200?500 mm (Fig. 2C). The colocalization of siRNA with GFAP-positive astrocytes indicated that glial cells were efficiently transfected by the fluorescence-labeled siRNA in the anterior funiculus at five days after SCI (Fig. 2B, 2D). Moreover, the results of immunostaining showed that the most evident reduction of GFAP and vimentin was achieved around the spinal contusion in the PMW group at five days after SCI (Fig. 4A, 4B, 4C, 4D). These observations are attributable to the capability of PMWs to deliver siRNAs into deeply located astrocytes in the spinal contusion. As described above, astrocytes are activated after SCI with enlarged somas and intensive expression of IF proteins over time, and then a cystic cavity is formed in the region surrounded by the glial scar [21]. This neurodegenerative nature leads to a progressive increase in the size of the cavitation area [76,77]. The results of immunohistological analysis at five days post-SCI showed that GFAP and vimentin were markedly up-regulated around the lesion in the SCI group, resulting in prominent formation of glial scars and cavities at three weeks after injury (Fig. 5A, 5B, 6A). For effective reduction of glial scar formation, itis necessary to deliver the relevant siRNAs into a broad region in the spinal contusion, and then to reduce astrogliosis. At five days post-SCI, the IF proteins were silenced especially in the white matter of the anterior horns in the PMW group. This shows that PMWs enhanced the uptake of siRNA into glial cells, especially those located in the ventral funiculus. The anterograde tracing experiment showed numerous regenerating CST axons in the vicinity of the lesion in the PMW group (Fig. 7). This demonstrates that inhibition of excessive glial activity in the injured spinal tissue causes promotion of spontaneous axonal outgrowth, leading to functional recovery. The motor function of the lower limbs of rats in the PMW group was significantly improved from five days after injury (Fig. 8). These findings are consistent with the results of a previous study in which double mutant mice lacking GFAP and vimentin showed significant axonal regrowth of descending fibers of the corticospinal tract and ventral horn serotonergic tract, leading to an improved functional recovery after spinal cord hemisection [20]. To apply PMW-based siRNA therapy to larger animals with a thicker spinal column, deeper propagation of PMWs and broader distribution of therapeutic siRNAs are required. In our recent study, significant gene expression was observed in rat skin as a test tissue by applying PMWs that had to first propagate through 15-mm-thick tissue 12926553 phantoms, demonstrating the capability of PMWs for cell permeabilization in tissues of ,15 mm.
