We investigated experimentally dependence of light fluence on treated depth with photosensitization reaction shortly
after photosensitizer injection in rabbit myocardial tissue in vivo. In this particular photosensitization reaction scheme,
the photosensitizer accumulation characteristics for target region are not available. Meanwhile, the photosensitizer dose
and hospitalization period under restricted light circumstance might be reduced. Since both photosensitizer and oxygen supply are governed by blood flow, this photosensitization reaction is influenced significantly by blood flow variation in
particular blood vessel occlusion. We employed the myocardial tissue to keep tissue blood flow during the
photosensitization reaction because vessel blood flow speed in myocardial tissue is fast to resist vascular occlusion.
Surgically exposed rabbits myocardial tissues were irradiated with the light fluence ranging 25-100 J/cm2 by a 663 nm
diode laser 30 min after the injection of 2 mg/kg water soluble chlorin photosensitizer, Talaporfin sodium. Two weeks
after the irradiation, the rabbits were sacrificed and the histological specimens of the irradiated area were made to
measure scar layer thickness. The scar layer tissue thickness of 0.2-3.0 mm was observed microscopically by the light
fluence ranging 25-100 J/cm2. The scarring threshold in the deposit light fluence was estimated to 15-25 J/cm3 based on the above mentioned relation assuming constant and uniform myocardial effective attenuation coefficient of 0.72 mm-1.
The estimated scarring threshold in the deposit light fluence was lower than the threshold of conventional PDT. Large variation of the estimated threshold value might be attributed to unconsidered PDT parameter such as flow rate inhomogeneity in the myocardial tissue. These results suggested that the photosensitization reaction investigated in this
study would be available to apply arrhythmia therapy such as atrial fibrillation.
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.
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.
We demonstrated a possibility of electrical conduction block by ex vivo and in vivo experiments using rat models to
establish a non-thermal treatment for atrial fibrillation by photosensitization reaction (PR). One hour after the
injection of 2 mg/kg Talaporfin sodium to Wistar rat, the right ventricle (1.4 mmT) was extracted. Paced with a
stimulation electrode, this tissue was placed in a tissue bath and immersed in irrigated Tyrode's solution of 37°C
with 8 μg/ml Talaporfin sodium and the gas mixture bubbling of 95% CO2 and 5% O2. The propagated electrical
signal was measured by two bipolar electrodes. Exciting light of 670 nm in wavelength was irradiated to the tissue
between the bipolar electrodes by the power density of 1 W/cm2. After this irradiation, propagation signal blockade
was obtained and continued up to three hours. Rat atrioventricular (AV) node was employed as a target region for
chronic model. The heart of Wistar rat was surgically exposed. External four-lead electrocardiogram of this rat was
measured. Thirty minutes after the injection of 10 mg/kg Talaporfin sodium to the rat, exciting light of 663 nm in
wavelength was irradiated to the AV node by the power density of 500 mW/cm2 for ten minutes. Acute AV block
was obtained during the irradiation. Two weeks after this procedure, complete AV block was confirmed. The rat
was sacrificed to obtain the tissue specimen. We found that the AV node was replaced by scarring tissue under the
microscopic observation of the specimen. We verified possibility of permanent electrical conduction block using PR.
We have proposed a new type of atrial fibrillation treatment with the early state photodynamic therapy (PDT), in
which the interval time between the photosensitizer injection and irradiation is shorter than that in conventional way.
We had demonstrated the acute electrical blockade by the PDT with talaporfin sodium and a red (670 nm) diode
laser in ex vivo and in vivo experiment using rat normal myocardial tissue. The previous study of intracellular Ca2+
concentration measurement in rat cardiac myocytes during the PDT indicated that Ca2+ influx induced by the plasma
membrane damage might be the main cause of the acute reaction of myocardial tissue. We found that the cell
damage of cardiac myocytes triggered by the PDT was mainly influenced by the site where the photosensitizer
exists. In this study, we examined the relationship between the sites of talaporfin sodium existing and cell death
phenotypes in response to the PDT, in order to clarify the mechanism of the acute electrical blockade induced by the
PDT in myocardial tissue. The talaporfin sodium fluorescence was observed after the various incubation times to
visualize the time-lapse intracellular photosensitizer localization. The distribution of the photosensitizer was
dependent on the incubation time. The change in intracellular Ca2+ concentration during the PDT was examined
with a fluorescent Ca2+ indicator by a high-speed Nipkow confocal laser microscope (CSU-X1, Yokogawa Electric
Company). We obtained the Ca2+ dynamics during the PDT which can explain the PDT-induced cell death pathways.
We concluded that the Ca2+ influx induced by plasma membrane damage is the possible mechanism of the electrical
blockade by the early state PDT.
We investigated the correlation between the therapeutic effect by early irradiation Photodynamic Therapy (PDT) and vascular response. The early irradiation PDT has been proposed by our group. This PDT protocol is that pulse laser irradiates to tumors 1 h after intravenous injection of water-soluble photosensitizer. The intact layer appeared over the well treated layer, when the early irradiation PDT was performed at rat prostate subcutaneous tumors with high intensity pulse laser (over 1 MW/cm2 in peak intensity) and Talaporfin sodium. In order to clarify the phenomenon mechanism, we monitored blood volume, surface temperature, photosensitizer amount, and oxygen saturation during the PDT. The rat prostate subcutaneous tumor was irradiated with excimer dye laser light at 1 h after the intravenous injection. The photosensitizer dose wa 2.0 mg/kg, and the pulse energy density was 2.5 mJ/cm2 (low intensity) or 10 mJ/cm2 (high intensity). Under the low intensity pulsed PDT, the fluorescence amount was decreasing gently during the irradiation, and the blood volume and oxygen saturation started decreasing just after the irradiation. Under the hgh intensity pulsed PDT, the fluorescence amount was decreaased rapidly for 20 s after the irradiation started. The blood volume and oxygen saturation were temporally decreased during the irradiation, and recovered at 48 hrs after the irradiation. According to these results, under the low intensity pulsed PDT, the blood vessel located near the surface started closing just after the irradiation. On the other hand, under the high intensity pulsed PDT the blood vessel was closing for 20 s after the irradiation started, moreover, the blood flow recovered at 48 hrs after the irradiation. We concluded that the vascular response depended on the pulse energy density, and then the therapeutic effect was attributed to the difference of the vascular response. In other words, the surface intact layer could be considered to be induced the temporal drug and oxygen depletion effect associated with the temporal vascular shutdown.
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