2.1.

Animal Preparation

Female Sprague-Dawley rats (Japan SLC) weighting 180 to 270 g were used in the present study under the protocol approved by the Committee on the Ethics of Animal Experiments in the National Defense Medical College. Before the operation, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg animal weight).

2.2.

Generation and Measurement of Pressure Characteristics of Photomechanical Waves

PMWs were generated by irradiating a target, a 0.5-mm-thick black natural rubber disk of 5 mm in diameter, with 6-ns (FWHM) laser pulses from the second harmonic wave (532-nm) of a Q-switched Nd:YAG laser (Brilliant b, Quantel, Les Ulis Cedex, France). The laser pulses were focused with a plano-convex lens (f = 200 mm) to a diameter of 3 mm on the rubber. A 1.0-mm-thick transparent polyethylene terephthalate sheet was bonded on the rubber to confine the laser-produced plasma to increase the peak pressure and impulse of the PMWs.^{32, 33}

Before the gene delivery experiment, the pressure profiles of PMWs were measured using a needle-type PZT hydrophone with a sensitive area of 1.0 mm in diameter (HNR-1000, Onda, Sunnyvale, California), based on the same experimental configuration as that described in our previous papers.^{25, 27, 28, 29, 34} Ultrasonic conductive gel (Echo Jelly, Aloka, Tokyo, Japan) was used to match the acoustic impedances of the laser target and the hydrophone protection film. The output signals were recorded by a digital oscilloscope (TDS680B, Tektronix, Inc., Beaverton, Oregon; 1 GHz bandwidth). The measurement was carried out three times for each laser irradiation condition.

2.3.

Construction of Plasmid DNA

Plasmid DNA encoding EGFP (pEGFP-C1, Palo Alto, California), which was driven by the cytomegalovirus (CMV) promoter, was purchased from Clontech. Plasmid DNA encoding firefly luciferase driven by the CMV promoter was provided by Dr. Kaneda.³⁵ The plasmids were transformed into Escherichia coli competent cells and then amplified and purified with a Qiagen column. The vector used as a control vector was a CMV vector plasmid not containing luciferase cDNA. The plasmid vectors were prepared at a final concentration of 2.0 μg/μl in a TE solution (10 mM Tris and 1 mM EDTA, pH 8.0).

2.4.

Photomechanical Wave-based Gene Transfer to the Spinal Cord

The spinal cord in a rat was exposed by removing the dorsal part of the tenth thoracic vertebra. A 27-gauge needle of a Hamilton syringe was stereotaxically inserted into the spinal cord at a point 1.0 mm distant from the midline and 2.0 mm in depth from the surface. The plasmid solution (10 μl) was slowly injected to minimize tissue damage in the spinal cord and a laser target was placed at the injection point on the spinal cord surface (Fig. 1). Ultrasonic conductive gel was used between the target bottom surface and the spinal surface to ensure acoustic impedance matching. The target was irradiated with a single laser pulse to generate a PMW. For multi-pulse irradiation, the target was replaced for each pulse to keep efficient confinement of the laser-produced plasma. For the experiment on EGFP gene transfer, three laser irradiation conditions to generate PMW(s) were examined: three pulses with a laser fluence of 0.3 J/cm², ten pulses with 0.3 J/cm², and one pulse with 0.9 J/cm².

Fig. 1

Configuration for gene transfer into the rat spinal cord by use of PMWs.

2.5.

Gene Expression Assay

At 48 h post-transfection, animals transfected with the EGFP gene were deeply anesthetized and perfused with 50 ml physiological saline and 200 ml of 4% paraformaldehyde. The spinal cord was removed, post-fixed in the same fixative as that used for perfusion for 16 h, and stored in 0.1 M phosphate-buffered saline for 24 h. The tissue was embedded in an optimal cutting temperature compound (Tissue-Tek) and then frozen and sectioned with a cryostat microtome to 10 μm in thickness to observe the fluorescence of EGFP, for which a fluorescent microscope (Eclipse E600, Nikon Corporation, Tokyo, Japan) was used. For the digital fluorescent images obtained, EGFP-derived green pixels were discriminated on the basis of RGB values. For semi-quantitative evaluation of gene expression, the total numbers of EGFP-derived green pixels in the white matter, and the gray matter were counted. Histological analyses were carried out for four rats in each gene transfer condition.

To investigate the targeting characteristics of PMW-based gene delivery into spinal tissue, we evaluated luciferase activity for the tissue in each spinal segment by using a standard luciferase assay kit (E1500, Promega Corporation, Madison, Wisconsin). The harvested spinal tissues were homogenized with fine surgical scissors in 500 μl lysis buffer solution. The homogenates were centrifuged at 15,000 rpm for 15 min at 4°C. The homogenate supernatant (20 μl) was added to 100 μl luciferin in a 5-ml polystyrene round bottom tube and the luminescence was measured for 10 s with a luminometer (Flash and Glow LB955, Berthold Technologies, Bad Wildbad, Germany). Analysis of luciferase activities in each spinal segment under the dorsal part of 8th to 12th thoracic vertebra was carried out at two days after gene transfer for five rats in each gene transfer condition. At one, two, five, and 10 days after application of PMWs, the spinal cord was harvested from the spinal segment under the dorsal part of 9th to 11th thoracic vertebra (n = 5).

2.6.

Evaluation of Locomotive Function

To evaluate the noninvasiveness of PMW-based gene transfer, hind limb movements at 24 h after the operation were observed in an open field and scored based on the locomotor BBB scale. The criteria for rating on the 21-point score were adopted from Basso;³¹ a score of zero means no spontaneous movement, while a score of 21 indicates normal locomotion. Evaluations were carried out for four to six rats in each EGFP gene transfer condition. For comparison, sham-treated animals were subjected to laminectomy but received neither weight-drop injury nor PMWs (n = 4).

2.7.

Statistical Analysis

Analyses of EGFP gene expression in the tissue and luciferase activities in each spinal segment were based on Kruskal-Wallis test with Scheffe's method for multiple comparisons. Comparisons of temporal changes in luciferase expression were performed using a nonparametric Mann-Whitney's U-test. Statistical analysis of locomotive functional evaluation was performed using Kruskal-Wallis test followed by Steel's method for multiple comparisons, including the sham control: laminectomy alone. A value of P < 0.05 was regarded as statistically significant in these analyses.

3.1.

Pressure Characteristics of Photomechanical Waves

Figure 2 shows pressure temporal profiles of PMWs used for gene transfer to rat spinal cords. The pressure waveforms were dominated by positive pressure, although a negative pressure component, which can cause cavitation, was observed at 0.3 J/cm². However, biological tissues are generally much less susceptible to compressive stress than to tensile stress. In addition, the pulse widths of the PMWs were as short as 170 ns (FWHM), resulting in relatively small values of impulse despite the high peak pressures. These characteristics would be associated with the low invasiveness of the present gene transfection. With laser fluences of 0.3 and 0.9 J/cm², the peak pressures were about 45 and 135 MPa, respectively; the rise times (rise time defined as the time from 10 to 90% of peak pressure) were about 42 and 34 ns, respectively. Thus, the stress gradient (peak pressure divided by rise time) with a laser fluence of 0.9 J/cm² (4.0 MPa/ns) was about 3.7 times higher than that with a laser fluence of 0.3 J/cm² (1.1 MPa/ns).

Fig. 2

Typical temporal profiles of PMWs at laser fluences of 0.3 and 0.9 J/cm² with a 3-mm spot diameter.

3.2.

Distributions of Enhanced Green Fluorescent Protein Gene Expression

Figure 3 shows fluorescence images of EGFP gene expression in sagittal sections of the rat spinal cords treated under the four different conditions at 48 h post-transfection. In the negative control section, for which the spinal cord was only injected with physiological saline, no fluorescence was observed (data not shown). For the rats with plasmid DNA injection only (no PMWs), EGFP expression was observed in the gray matter with almost no expression in the white matter [Fig. 3a]. For the rats with PMW application after plasmid injection, on the other hand, EGFP fluorescence was observed in both the gray and white matters [Figs. 3b, 3c, 3d]. Figure 4 shows the results of semi-quantitative evaluation of EGFP expression in the sections, for which the number of pixels showing green fluorescence was counted for each image. Pixels showing fluorescence of lower intensity than the background level (fluorescence intensity level in the negative control) were excluded in this evaluation. The level of EGFP gene expression in the white matter was higher for all of the PMW application groups than for the group with plasmid injection only, suggesting that PMW-based gene transfection is more effective for cells in the white matter than for those in the gray matter. However, the type of cells showing EGFP gene expression, e.g., neurons or glial cells, was not determined in the present study.

Fig. 3

Expression of EGFP gene in the rat spinal cord under the four different treatment conditions: (a) plasmid DNA injection only; (b), (c), (d) PMW application after plasmid injection. (b) 0.3 J/cm² × 3 pulses, (c) 0.3 J/cm² × 10 pulses, and (d) 0.9 J/cm² × 1 pulse. Broken lines indicate the boundary between the white (upper) and the gray (lower) matters. Scale bars indicate 200 μm.

Fig. 4

Number of pixels showing green fluorescence in the EGFP expression images of rat spinal cords under the four different treatment conditions: (a) plasmid DNA injection only; (b), (c), (d) PMW application after plasmid injection. (b) 0.3 J/cm² × 3 pulses, (c) 0.3 J/cm² × 10 pulses, and (d) 0.9 J/cm² × 1 pulse. Values are expressed as means + S.E.M (n = 4). ** depicts P < 0.01 versus plasmid injection only.

The expression level would be dependent of the laser irradiation conditions. In the white matter, a significantly higher EGFP gene expression level was observed with a laser fluence of 0.3 J/cm² and a pulse number of 10 than that in the control group, although there was no significant difference in gene expression level in the gray matter among the groups. The highest averaged EGFP expression level was observed in spinal tissue exposed to 10 pulses of PMWs generated at a laser fluence of 0.3 J/cm², the total laser energy being highest in all of the groups. In the two groups exposed to the same total laser energy of 0.9 J/cm² (0.3 J/cm² × 3 pulses and 0.9 J/cm² × 1 pulse), there was no significant difference between gene expression levels in the white matter or between gene expression levels in the gray matter. Based on these results, we employed 10 pulses of PMWs generated at 0.3 J/cm² as the optimum laser irradiation conditions in the following experiments.

3.3.

Targeting Characteristics

Figure 5 shows expression levels of luciferase gene expressed as relative light units (RLU) per milligram of tissue protein for each thoracic spinal segment at two days after gene transfer. In this experiment, plasmid DNA was stereotaxically injected into the spinal cord under the 10th thoracic vertebra (Th 10). The average length and width of the Th-10 vertebra was approximately 6 and 5 mm, respectively. The negative control (control vector injection) shows luciferase activity in the spinal cord injected with the control vector without PMW application (background level of luciferase activity), which was less than 10² RLU per milligram of tissue protein. For Th 10, the level of luciferase activity obtained by PMW application after plasmid DNA injection (PMW application group) was significantly higher (by 16-fold) than that of the negative control. The level of luciferase activity obtained by plasmid DNA injection alone (plasmid DNA injection alone group) was also higher than that of the negative control, but there was no significant difference. For Th 9 and Th 11, the level in the PMW application group was also higher than the levels in other groups, but there were no significant differences.

Fig. 5

Levels of luciferase gene expressions for each thoracic (Th) spinal segment (approximately 6 mm in length) under the three different treatment conditions: control vector injection alone, plasmid DNA injection alone and PMW application after plasmid DNA injection. Control vector or plasmid DNA was injected into the spinal cord segment under the 10th thoracic vertebra, to which PMWs were applied. Results are expressed as means + S.E.M (n = 5). ** depicts P < 0.01.

Figure 6 shows time courses of luciferase gene expression levels integrated for Th 9, Th 10, and Th 11, where the level before any treatment is shown for comparison. Luciferase activity decreased with elapse of time both in the PMW application group and the plasmid injection alone group. At two and five days after gene transfection, however, the luciferase activity levels in the PMW application group were significantly higher than those in the plasmid injection alone group. For the plasmid injection alone group, the level of luciferase activity at five days after plasmid injection was comparable to that before gene transfection. At 10 days after gene transfection, the luciferase expression levels in both groups were similar to that before gene transfection.

Fig. 6

Time courses of luciferase gene expressions integrated for the 9th, 10th, and 11th thoracic spinal segments for the plasmid injection alone group and the PMW application group. The luciferase activity before treatment is also shown for comparison. Values are expressed as means +S.E.M (n = 5 to 10). * depicts P < 0.05.

3.4.

Locomotive Function

Locomotive damage to the spinal cord after PMW-based gene transfer was examined based on the BBB scores. Figure 7 shows BBB scores for the rats at 24 h after EGFP gene transfer. The averaged locomotive score in the plasmid injection alone group was 20.3, which was nearly the same as the score in the sham operation group (20.9, not shown Fig. 7). This indicates that intrathecal injection was noninvasively conducted in the present experiments. There was also no significant difference in BBB scores between the group with naked plasmid DNA injection alone and the two groups with PMW application (0.3 J/cm² × 3 pulses and 0.3 J/cm² × 10 pulses), the averaged scores in these three groups being 20.0, 20.0, and 20.1, receptively. For the irradiation condition of 0.9 J/cm² × 1 pulse, on the other hand, the averaged score was significantly low (12.6) compared to that in the plasmid injection alone group.

Fig. 7

Results of functional evaluation based on BBB scoring at 24 h after EGFP gene transfer under the same four treatment conditions as those shown in Figs. 3 and 4. Values are expressed as means + S.D (n = 4 to 6). * depicts P < 0.05.