Ays before dissection (n = 4).Histological AnalysisIn the same Benzocaine site manner as described above, frozen sagittal serial sections (thickness of 10 mm) were prepared using a cryostat microtome after animal sacrifice at five days (n = 3, each group) or twenty-one days (n = 3, each group) after SCI. For diaminobenzidine (DAB) staining, after blocking of endogenous peroxidase with 3 hydrogen peroxide, the sections were incubated overnight at 4uC with rabbit polyclonal anti-GFAP (1:3; N1506, Dako, Tokyo, Japan) or with mouse monoclonal anti-vimentin (1:10; sc-6260,Treatment of SCI by PMW-Mediated siRNA DeliverySanta Cruz Biotechnology, CA). After washing with Tween-PBS, the slices on slide glasses were incubated with secondary antibody linked with dextran polymer peroxidase complex for thirty minutes at room temperature. Peroxidase-based color development was performed using 3, 3-diaminobenzidine. For evaluation of the morphological changes at twenty-one days after SCI, the adjacent sections were also stained with hematoxylin and eosin (HE). For a typical histological image at the lesion epicenter and adjacent sites (n = 3 for the same animal), cavitation areas in the longitudinal images of HE staining were manually outlined and quantified by image analysis (n = 9, each group).Anterograde LabelingRegeneration of corticospinal tracts (CSTs) was assessed using an anterograde tracer (mini-ruby) injected into the motor cortices. Two weeks after SCI, animals were anesthetized with pentobarbital sodium and two holes of 1.0 mm in diameter were drilled in the cranium to expose both motor cortices (coordinates: 2 mm posterior to the bregma, 2 mm lateral to the bregma) (n = 4, each group) [47?0]. A 35-gauge needle of a Hamilton syringe was inserted through each hole to a depth of 1.0?.5 mm and five injections were made for each side; each injection consisted of 1.0 ml of 10 tetramethylrhodamine biotinylated dextran (miniruby, Molecular Probes, Eugene, OR). The needle was retained in place for 2 minutes after completion of the injection to allow diffusion of the dextran. One week after the injection, the rats were perfused with 4 paraformaldehyde in physiological saline. In the same manner as that described above, frozen sections (thickness of 25 mm) were prepared using a cryostat microtome. The longitudinal sections at the lesion epicenter were examined under a fluorescence microscope (Axiovert 200, Carl Zeiss, Thornwood, NY).after propagation through an extracted spinal cord were measured using a 370-86-5 hydrophone under the conditions used for siRNA transfection (Fig. 1A). The waveforms both before and after propagation through the tissue were dominated by a compressive pressure component, suggesting low invasiveness of PMWs to the tissue (Fig. 1B) because biological tissues are generally much less susceptible to compressive stress than to tensile stress [51]. The maximum pressures at the spinal cord surface and the bottom were about 51 MPa and 20 MPa, respectively. The pulse widths of PMWs were as short as 80?00 ns (full width at half maximum, FWHM). Thus, the impulses of PMWs were relatively small despite the high peak pressures, which were 7.3 Pa?s and 1.8 Pa?s at the spinal cord surface and the bottom, respectively.Delivery of Fluorescence-labeled siRNAs into Injured Spinal Cords by PMWsFluorescence-labeled siRNAs were used to examine the distribution of siRNAs delivered by applying PMWs to injured spinal cord. Immediately after making a contusion in a rat s.Ays before dissection (n = 4).Histological AnalysisIn the same manner as described above, frozen sagittal serial sections (thickness of 10 mm) were prepared using a cryostat microtome after animal sacrifice at five days (n = 3, each group) or twenty-one days (n = 3, each group) after SCI. For diaminobenzidine (DAB) staining, after blocking of endogenous peroxidase with 3 hydrogen peroxide, the sections were incubated overnight at 4uC with rabbit polyclonal anti-GFAP (1:3; N1506, Dako, Tokyo, Japan) or with mouse monoclonal anti-vimentin (1:10; sc-6260,Treatment of SCI by PMW-Mediated siRNA DeliverySanta Cruz Biotechnology, CA). After washing with Tween-PBS, the slices on slide glasses were incubated with secondary antibody linked with dextran polymer peroxidase complex for thirty minutes at room temperature. Peroxidase-based color development was performed using 3, 3-diaminobenzidine. For evaluation of the morphological changes at twenty-one days after SCI, the adjacent sections were also stained with hematoxylin and eosin (HE). For a typical histological image at the lesion epicenter and adjacent sites (n = 3 for the same animal), cavitation areas in the longitudinal images of HE staining were manually outlined and quantified by image analysis (n = 9, each group).Anterograde LabelingRegeneration of corticospinal tracts (CSTs) was assessed using an anterograde tracer (mini-ruby) injected into the motor cortices. Two weeks after SCI, animals were anesthetized with pentobarbital sodium and two holes of 1.0 mm in diameter were drilled in the cranium to expose both motor cortices (coordinates: 2 mm posterior to the bregma, 2 mm lateral to the bregma) (n = 4, each group) [47?0]. A 35-gauge needle of a Hamilton syringe was inserted through each hole to a depth of 1.0?.5 mm and five injections were made for each side; each injection consisted of 1.0 ml of 10 tetramethylrhodamine biotinylated dextran (miniruby, Molecular Probes, Eugene, OR). The needle was retained in place for 2 minutes after completion of the injection to allow diffusion of the dextran. One week after the injection, the rats were perfused with 4 paraformaldehyde in physiological saline. In the same manner as that described above, frozen sections (thickness of 25 mm) were prepared using a cryostat microtome. The longitudinal sections at the lesion epicenter were examined under a fluorescence microscope (Axiovert 200, Carl Zeiss, Thornwood, NY).after propagation through an extracted spinal cord were measured using a hydrophone under the conditions used for siRNA transfection (Fig. 1A). The waveforms both before and after propagation through the tissue were dominated by a compressive pressure component, suggesting low invasiveness of PMWs to the tissue (Fig. 1B) because biological tissues are generally much less susceptible to compressive stress than to tensile stress [51]. The maximum pressures at the spinal cord surface and the bottom were about 51 MPa and 20 MPa, respectively. The pulse widths of PMWs were as short as 80?00 ns (full width at half maximum, FWHM). Thus, the impulses of PMWs were relatively small despite the high peak pressures, which were 7.3 Pa?s and 1.8 Pa?s at the spinal cord surface and the bottom, respectively.Delivery of Fluorescence-labeled siRNAs into Injured Spinal Cords by PMWsFluorescence-labeled siRNAs were used to examine the distribution of siRNAs delivered by applying PMWs to injured spinal cord. Immediately after making a contusion in a rat s.
