Tsunenori Arai

The Myocardial Electrical Blockade Induced by Photosensitization Reaction

The authors studied the application of photosensitization-reaction-induced cytotoxicity to establish electrical blockade of myocardial tissue. This photosensitization-reaction-induced cytotoxicity, i.e., photodynamic therapy (PDT) was performed with chlorine photosensitizer, talaporfin sodium, and a red (670 nm) diode laser. The cytotoxicity on rat cardiac myocytes and the electrical blockade by PDT using rat myocardial tissue were confirmed. The mechanism of PDT-induced electrical blockade was investigated. The photosensitization-reaction-induced cytotoxicity in normal rat cardiac myocytes was obtained in cell lethality measurement. The ex vivo experiment with rat-isolated myocardial tissue demonstrated the immediate electrical blockade by PDT. Moreover, the possibility of permanent electrical blockade by PDT using rat atrioventricular blockade model was confirmed. To study the mechanism of the acute electrical blockade obtained in the ex vivo study, intracellular Ca2+ concentration changes in rat cardiac myocytes were measured by the intensity of the fluorescent Ca2+ indicator Fluo-4 AM. A rapid increase in fluorescence intensity during the photosensitization reaction and a change in cell morphology after the photosensitization reaction were observed. These results indicate that cell membrane damage, Ca2+ influx, and eventually cell death are caused by the photosensitization reaction. The necrosis-like cell death induced by the photosensitization reaction can explain a permanent electrical blockade of the myocardial tissue in vivo by PDT.

Myocardial electrical conduction block induced by photosensitization reaction in exposed porcine hearts in vivo

This study proposes photosensitization reaction for non‐thermal cardiac ablation in arrhythmia therapy. Acute and chronic phase experiments were conducted in exposed porcine hearts to demonstrate the photosensitization reaction‐induced myocardial electrical conduction block in vivo.

Photosensitization reaction-induced acute electrophysiological cell response of rat myocardial cells in short loading periods of talaporfin sodium or porfimer sodium

Electrophysiological responses of rat myocardial cells to exogenous photosensitization reactions for a short period of incubation with two photosensitizers, talaporfin sodium or porfimer sodium, were measured in a subsecond time scale. The loading period of the photosensitizer when the photosensitizer might not be taken up by the cells was selected as 15u2003min, which was determined by the fluorescence microscopic observation. We measured the intracellular Ca2+ concentration ([Ca2+]in) by using a fluorescent Ca2+ indicator, Fluo‐4 AM, under a high‐speed confocal laser microscope to evaluate the acute electrophysiological cell response to the photosensitization reaction. The measured temporal change in Fluo‐4 fluorescence intensity indicated that the response to the photosensitization reaction might be divided into two phases in both photosensitizers. The first phase is acute response: disappearance of Ca2+ oscillation when irradiation starts, which might be caused by ion channel dysfunction. The second phase is slow response: [Ca2+]in elevation indicating influx of Ca2+ due to the concentration gradient. The continuous Ca2+ influx followed by changes in cell morphology suggested micropore formation on the surface of the cell membrane, resulting in necrotic cell death.

Basic study of charring detection at the laser catheter-tip using back scattering light measurement during therapeutic laser irradiation in blood

The purpose of this study is to investigate transient process of the charring at the laser catheter-tip in blood during therapeutic laser irradiation by the back scattering light measurement to detect precursor state of the charring. We took account of using photodynamic therapy for arrhythmia in blood through the laser catheter. We observed the influence of the red laser irradiation (λ=663 nm) upon the shape of red blood cells (RBCs). The RBCs aggregation, round formation, and hemolysis were took place sequentially before charring. With a model blood sandwiched between glass plates simulated as a catheter-tip boundary, we measured diffuse-reflected-light power and transmitted-light power simultaneously and continuously by a microscopic optics during the laser irradiation. We found that measured light power changes were originated with RBCs shape change induced by temperature rise due to the laser irradiation. A gentle peak following a slow descending was observed in the diffuse-reflected-light power history. This history might indicate the precursor state of the charring, in which the hemolysis might be considered to advance rapidly. We think that the measurement of diffuse-reflected-light power history might be able to detect precursor state of charring at the catheter-tip in blood.

Detection of pre-charring optical behavior at a laser catheter-tip in blood: Ex vivo and in vivo study

We studied a pre-charring optical behavior of blood at a laser catheter-tip during a red laser irradiation (663 nm, CW) with around 50 W/cm2 in blood to prevent charring at the laser catheter-tip. The laser irradiated red-blood-cell shape changes were microscopically observed. A round formation, aggregation, and hemolysis were found until blood charring (ex vivo). A time-history of diffuse-reflected light power and transmitted light power from a thin blood layer which was irradiated by the red laser were measured with microscope optics to investigate the charring process. The diffusereflected light power decreased following a gentle peak before the charring. This decrease indicated the pre-charring behavior which might be induced by scattering and absorption changes due to red-blood-cell degenerations described above. Using the laser catheter located in porcine heart, we successfully detected the pre-charring behavior by a backscattering light power (in vivo). We demonstrated charring prevention availability with the laser power control (ex vivo). We think that the backscattering light power measurement and laser power control via the laser catheter might be useful to detect pre-charring behavior, and to prevent the charring for therapeutic laser irradiation in blood under catheterization such as arrhythmia treatment with photodynamic therapy.

The new application of photosensitization reaction to atrial fibrillation treatment: mechanism and demonstration of non-thermal electrical conduction block with porcine heart

We have proposed non-thermal electrical conduction block for atrial fibrillation treatment by the photosensitization reaction, in which the interval time between the photosensitizer injection and irradiation is less than tenth of that in conventional way. To study the mechanism of photosensitization reaction-induced electrical conduction block, intracellular Ca2+ concentration change in rat myocardial cells was measured by fluorescent Ca2+ indicator Fluo-4 AM with confocal laser microscopy. Measured rapid increase in the fluorescence intensity and a change in cell morphology indicated that cell membrane damage; that is Ca2+ influx and eventually cell death caused by the photosensitization reaction. To demonstrate myocardial electrical conduction block induced by the photosensitization reaction, surgically exposed porcine heart under deep anesthesia was used. The myocardial tissue was paced with a stimulation electrode. The propagated electrical signals were measured by bipolar electrodes at two different positions. Thirty minutes after the injection of 5-10 mg/kg Porfimer sodium or Talaporfin sodium, the red laser light was irradiated to the tissue point by point crossing the measuring positions by the total energy density of less than 200 J/cm2. The electrical signal conduction between the measuring electrodes in the myocardial tissue was delayed by each irradiation procedure. The electrical conduction delay corresponded to the block line length was obtained. These results demonstrated the possibility of non-thermal electrical conduction block for atrial fibrillation treatment by the photosensitization reaction.

Non-thermal ablation technology for arrhythmia therapy: acute and chronic electrical conduction block with photosensitization reaction

We have examined the possibility of non-thermal ablation technology for arrhythmia therapy with photosensitization reaction, in which photochemically generated singlet molecular oxygen may induce myocardial electrical conduction block. In the most popular energy source for arrhythmia catheter ablation; radiofrequency current, the thermal tissue injury causes electrophysiological disruption resulting in electrical isolation of ectopic beats. The temperature-mediated tissue disruption is difficult to control because the tissue temperature is determined by the heating and thermal conduction process, so that severe complications due to excessive heat generation have been the problem in this ablation. We demonstrated the electrical conduction block of surgically exposed porcine heart tissue in vivo with photosensitization reaction. The acute myocardial electrical conduction block was examined by the stimulation and propagation set-up consisting of a stimulation electrode and two bipolar measurement electrodes. Fifteen to thirty minutes after the injection of 5-10 mg/kg water-soluble chlorine photosensitizer, Talaporfin sodium (NPe6, LS11), the laser light at the wavelength of 663 nm with the total energy density of 50-200 J/cm2 was irradiated several times with 3- 7 mm in spot-size to make electrical block line in myocardial tissue across the conduction pathway between the bipolar measurement electrodes. The propagation delay time of the potential waveform increased with increasing the irradiated line length. The observation of Azan-stained specimens in the irradiated area two weeks after the procedure showed that the normal tissue was replaced to the scar tissue, which might become to be permanent tissue insulation. These results demonstrated the possibility of non-thermal electrical conduction block for arrhythmia therapy by the photosensitization reaction.

Fundamental study on photodynamic therapy for atrial fibrillation: Effect of photosensitization reaction parameters on myocardial necrosis in vitro

We studied necrotic cell death effect on myocardial cells with photosensitizer existed outside the cells varying photosensitization reaction parameters widely in vitro. We have developed non-thermal ablator with the application of photosensitization reaction for atrial fibrillation. Since laser irradiation is applied shortly after photosensitizer injection, the photosensitization reaction is induced outside the cells. The interaction for the myocardial cells by the photosensitization reaction is not well understood yet on various photosensitization reaction parameters. Rat myocardial cells were cultured in 96 well plates for 7 days. The photosensitization reaction was applied with talaporfin sodium (NPe6) and the semiconductor laser of 663nm wavelength. The average drug light interval was set 8 mins. The photosensitizer concentration and radiant exposure were varied from 5 to 40 μg/ml and 1.2 to 60 J/cm2, respectively. The well bottom was irradiated by the red laser with irradiance of 293 mW/cm2. The photosensitizer fluorescence was monitored during the photosensitization reaction. Alive cell rate was measured by WST assay after 2 hours from the irradiation. In the case of the photosensitizer concentration of 10 μg/ml, the myocardial cells were almost alive even thought 60 J/cm2 in the radiant exposure was applied. In the 15 μg/ml case, the alive cell rate was almost linear relation to the photosensitizer concentration and radiant exposure. We obtained that the threshold for myocardial cell necrosis on the photosensitizer concentration was around 15 μg/ml with 20 J/cm2 in the radiant exposure. This threshold on the photosensitizer concentration was similar to the reported threshold for cancer therapy.

Optical property change of blood on an optical window boundary by 660-nm band laser irradiation

We studied an optical interaction on an optical window and blood boundary during the CW laser irradiation in 660 nm band until blood charring occurrence. We previously reported that a pre-charring optical behavior may be detected by diffuse-reflected-light power time-history. The aim of this study is to measure absorption coefficient (μa) and reduced scattering coefficient (μs) of a blood model to explain this pre-charring optical behavior. The blood model sandwiched between 2 glass slides to simulate the interface between blood and the optical window was used. A double integrating sphere system was constructed. The red laser in 660 nm band was irradiated to the sandwiched blood model. Fourty W/cm2 in irradiance was used as the maximum irradiance during irradiation via the laser catheter in vivo. μa and μs in the irradiated laser wavelength were measured continuously until blood charring occurrence using inverse adding doubling analysis. Continuous μa increase of 5-10% from the initial value until charring was observed. Decrease of μs with 8-10% during 15-30 s before charring following broad peak was obtained. We think these μa and μs changes may explain the pre-charring optical behavior detected by the diffuse-reflected-light power time-history in our reported study.

Basic study of effects on the smooth muscle cells' proliferation with novel short-term thermal angioplasty in vitro and in vivo

We investigated the effect on smooth muscle cells proliferation with stretch-fixing in both in vitro and in vivo porcine study to determine the optimum heat condition of novel short-term thermal angioplasty, Photo-thermo Dynamic Balloon Angioplasty (PTDBA). With PTDBA, we have obtained the sufficient arterial dilatation by short-term heating (< 15 s, < 70 °C) and low dilatation pressure (< 0.4 MPa) without excessive neo-intimal hyperplasia on chronic phase. The smooth muscle cells were found to be fixed with stretched shape in vascular wall after PTDBA in vivo. The deformation rate of smooth muscle cells nuclei was 1.6 ± 0.1 after PTDBA (15 s, 65 °C, 0.35 MPa). The smooth muscle cells, which were extracted from porcine arteries, were cultured on the specially designed equipment to give stretch-fixing stimulus in vitro. The cell proliferation was inhibited at 20 % stretching compared to 15 % stretching significantly (p < 0.05). The immunostaining specimens of basic Fibroblast Growth Factor (bFGF) and its receptor FGFR-1 were made from the porcine arteries in vivo. We found that the expressions of bFGF and FGFR-1 in the media were not observed after PTDBA. We think that these results suggested the possibility for the inhibition of the excessive cell proliferation after PTDBA.