Hitoshi Wakisaka

Gene transfer into mammalian cells by use of a nanosecond pulsed laser-induced stress wave

Plasmid DNA has been successfully delivered to mammalian cells by applying a nanosecond pulsed laser-induced stress wave (LISW). Cells exposed to a LISW were selectively transfected with plasmids coding for green fluorescent protein. It was also shown that transient, mild cellular heating (approximately 43 degrees C) was effective in improving the transfection efficiency.

Telepsychiatry: assessment of televideo psychiatric interview reliability with present‐ and next‐generation internet infrastructures

Objective: We assessed the reliability of remote video psychiatric interviews conducted via the internet using narrow and broad bandwidths.

High nitric oxide synthase activity in endothelial cells in ulcerative colitis.

Endothelial nitric oxide (NO) synthase, a unique NO synthase (NOS) isoform that is expressed constitutively by the vascular endothelium both in vivo and in vitro, is believed to be essential to systemic and/or local vascular integrity. NOS expression by endothelial cells may indicate vascular activation. We successfully established a simple method for the culture of microvascular endothelial cells from a small amount of tissue and investigated ulcerative colitis (UC), in which condition vascular factors have not been studied extensively. We cultured endothelial cells from the mesenteries of surgical patients with UC and assayed NOS activity by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Strong NOS activity was demonstrated in the cells from all UC patients (5/5), whereas no activity was detected in the cells from human umbilical veins and the mesenteries of colon cancer patients (0/10 and 0/5, respectively). This strong NOS activity was not diminished by incubation with a high concentration of glucocorticoid, suggesting that it was constitutive. These results indicate a close relationship of vascular activation (high NOS activity) with the pathogenesis of UC.

Biomedical engineering's contribution to defending the homeland

Reports on technological and biomedical initiatives being taken in Japan to counter bioterrorism. These include: telemedicine; noncontact vital sign monitoring using microwave radar; biomedical optical imaging of trauma diagnostics; antimicrobial photodynamic therapy; and biological weapon response.

Transdermal Delivery of Photosensitizer by the Laser-Induced Stress Wave in Combination with Skin Heating

To investigate the effect of combining skin heating with a laser-induced stress wave (LISW) on the drug permeability of the skin, we attempted to deliver porfimer sodium (Photofrin II) into rat skin in vivo. We demonstrated that a mild heating (~ 47°C) of the skin before applying the LISW enhanced the permeability of the drug into the skin. We suggest that the heating can increase the fluidity of the intercellular lipids and can cause swelling of the corneocytes, and therefore the LISW can easily form channels for drug transportation within the stratum corneum.

Delivery of Photosensitizer to Cells by the Stress Wave Induced by a Single Nanosecond Laser Pulse

We demonstrated that porfimer sodium (Photofrin II) was delivered into cells by applying a single nanosecond pulsed laser-induced stress wave (LISW). Analysis with a confocal laser scanning microscope showed that Photofrin molecules were homogeneously distributed in the cytoplasm at a low fluence (0.25 J/cm2), while the molecules were accumulated in a specific site at a high fluence (0.50 J/cm2). We also observed the nucleus membrane permeabilization induced by the LISW.

Channeling in myocardium tissue through nanosecond pulsed laser photodisruption at visible and near-infrared wavelengths

We investigated in vitro the mechanism of myocardium tissue ablation with nanosecond pulsed laser at the visible and near-infrared wavelengths. In experiments, porcine myocardium tissue was used as sample. It was found that the ablation rate at 1064 nm was larger than that at 532 nm in spite of lower absorption coefficient at 1064 nm than that at 532 nm for the tissue. During ablation the laser-induced optical emission intensity was measured and it was correlated with the ablation depth. Ablated tissue samples were fixed and stained, and histological analysis was performed with an optical microscope and a polarization microscope. For the 1064-nm laser-ablated tissues thermal damage was very limited, although damage that was presumably caused by mechanical effect was observed. The optical emission intensity during the 1064-nm laser ablation was higher than that during the 532-nm laser ablation at the same laser intensity. And for the 1064-nm laser ablation the ablation threshold was nearly equivalent to the optical emission. Based on these experimental results, we concluded that with the 1064-nm laser light, the tissue removal was achieved through a photodisruption process. Application of 1064-nm, nanosecond pulsed laser photodisruption to transmyocardial laser revascularization (TMLR) was discussed.

Characteristics and Mechanism of Myocardium Tissue Laser Ablation: In-vitro Study for Transmyocardial Laser Revascularization

To investigate the optimum laser condition for transmyocardial laser revascularization, ablation characteristics of porcine myocardium tissue have been measured in vitro in the spectral region from UV to near IR (230 - 1064 nm). In experiments, a nanosecond, tunable optical parametric oscillator and a Q-switched Nd:YAG laser (the fundamental band and its 2nd, 3rd, and 4th harmonics) were used for ablation. Measurement of ablation hole depth and diameter, and histological analysis with an optical microscopy were performed. It was found that the ablation efficiency increased and the thermal damage threshold of the tissue decreased with decreasing the wavelength. However, at relatively high fluences (approximately 5 J/cm2), unexpected deep ablation was obtained with a 1064-nm laser light, where not thermal but acoustic damage was observed near the walls of the ablation holes. Because intense laser- induced plasma was observed in this case, the shock wave would contribute to removal and/or damage of the tissue.

In vitro gene transfer by the use of laser-induced stress wave: Effect of laser irradiation conditions on transfection efficiency

Laser-mediated gene transfection is very attractive as a new method for targeted gene therapy because of its high spatial controllability of laser energy. Previously, we demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISW). In this study, we investigated the experimental conditions to increase transfection efficiency in vitro. By applying single-pulse LISW, transfection efficiency was increased with increasing laser fluence. Increase in the number of laser pulses increased transfection efficiency for laser fluences up to 1.3 J/cm2, but at higher fluences (>1.7 J/cm2), efficiency showed saturation tendency. Temperature dependence of transfection efficiency was also investigated.

In vitro gene transfer by the application of laser-induced stress wave: effect of cellular heating

We previously demonstrated in vitro that the simultaneous application of cellular heating and a laser-induced stress wave (LISW) enhanced the uptake of porfimer sodium (Photofrin) by cells. In this study, we attempted to apply this technique to gene transfer to cultured cells. LISW and/or a transient mild (~43°C) heating been applied to deliver plasmid coding for green fluorescent protein (EGFP) to NIH-3T3 cells. It was found that simultaneous application of an LISW and the heating significantly increased the transfection efficiency by a factor of 2.5 when compared with that for the cells treated with an LISW alone.