We experimentally studied the correlation between myocardial damage depth due to the extracellular photosensitization
reaction (PR) using talaporfin sodium and fluorescence-fall amount (FA), which is calculated from the measured
backscattering fluorescence intensity via a manipulatable 7 Fr. laser catheter during the PR operation in vivo to establish
treatment depth predictor for a non-thermal tachyarrhythmia treatment. The PR was performed to left and/or right
ventricle in the open-chest canine heart. The laser irradiation of 663±2 nm in wavelength via the laser catheter was
operated 15 min after the intravenous administration of talaporfin sodium with concentration of 36.2±8.0 μg/ml in plasma. The irradiation was operated with irradiance of 5, 10, 20 W/cm2, and duration of 5, 10, 20 s. Backscattering
fluorescence of 710±2 nm in wavelength was measured via the laser catheter during the PR. The FA was calculated
multiplying the irradiation duration by the fluorescence-fall, which is subtraction of the fluorescence intensity at the
kickoff and end of the irradiation. The canine heart was extracted 1 week after the PR and HE stained specimen was
histologically evaluated. The correlation of the myocardial damage depth and FA was investigated. We found that FA
obtained a logarithmic relation to the myocardial damage depth. We think that the FA might be available to predict the
PR induced myocardial damage depth for the application of tachyarrhythmia treatment under catheterization in vivo.
We studied the immediate response of myocardial cells by continuous observation using confocal microscope against oxidation stress by extracellular photosensitization reaction using Talaporfin sodium for tachyarrhythmia treatment application. Immediate response in order from several seconds to several minutes is required for the arrhythmia treatment since operators should judge the therapeutic effect during the tachyarrhythmia ablation procedure. To understand the immediate response of myocardial cells, we measured the intracellular Ca2+ concentration using fluo-4 AM during and after the extracellular photosensitization reaction. Talaporfin sodium concentration was varied 10-30 μg/ml. A red diode laser of 663 nm in wavelength was irradiated under the microscope with the radiant exposure of 40 J/cm2 and irradiance of 0.29 W/cm2. We observed the fluorescence image of fluo-4 AM each 400 ms during until 10 min after the photosensitization reaction. The myocardial cell beatings were stopped about 2 s after the beginning of the laser irradiation. The blebs were formed with the Ca2+ inflow. The intracellular Ca2+ was re-decreased after the bleb formation and then the cell necrosis was induced. The cell lethality 10 min after the laser irradiation was less than bleb formation ratio. The time response of the cell necrosis was shortened with the photosensitizer concentration increasing and the minimum average value was 209 s in the case of the 30 μg/ml in photosensitizer concentration and 40 J/cm2 in the radiant exposure. We think this extracellular photosensitization reaction may be applicable to tachyarrhythmia treatment in terms of its immediate response.
We investigated detailed extracellular photosensitization reaction effect on rat myocardial cells and the photosensitization reaction progress in a well to study a new application of photodynamic therapy for arrhythmia therapy.
We investigated electrophysiological and histological effect on canine anatomical isthmus in right atrium by photosensitization reaction (PR) of talaporfin sodium operated via a manipulative 7 Fr. laser catheter to establish a nonthermal tachyarrhythmia treatment. We continuously administrated talaporfin sodium via a left femoral vein to maintain photosensitizer concentration of 25-35 μg/ml in blood plasma, which is within the range of clinical use in human. Fifteen-minute after kickoff of the photosensitizer administration, a 663 nm laser was irradiated via the laser catheter for 30 s/point with irradiance of 10 W/cm2. After 17 times irradiations, a 23 ms delay of the electrical signal propagation along tricuspid annulus was observed. This result might demonstrate the acute electrical conduction delay induced by PR. The canine heart was extracted 10 days after PR and Azan staining specimen was histologically evaluated to investigate the myocardial damage by PR. The transmural fibrosis in anatomical isthmus was found. We demonstrated the PR-induced electrical conduction delay in acute phase in vivo using continuous photosensitizer dosed canine model, which maintain the clinical photosensitizer concentrat
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 (μ's) 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 μ's 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 μ's
with 8-10% during 15-30 s before charring following broad peak was obtained. We think these μa and μ's changes may
explain the pre-charring optical behavior detected by the
diffuse-reflected-light power time-history in our reported study.
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.
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.
We have studied a new type of myocardial catheter ablation with photosensitization reaction to realize non-thermal
therapy for atrial arrhythmia, such as atrial fibrillation. Photochemically-generated reactive oxygen species may induce
myocardial electrophysiological damage without heat generation. In this study, to demonstrate photosensitization
reaction-induced myocardial electrical conduction block, the inferior vena cava to tricuspid annulus (IVC-TA) isthmus
linear ablation was conducted with photosensitization reaction in porcine heart in vivo, using a newly developed laser
catheter (7 Fr.). The end point of the procedure was the production of IVC-TA isthmus block under the
electrophysiological analysis by diagnostic catheter with 10-bipole electrodes placed in right atrium along the isthmus.
Talaporfin sodium (NPe6) as a photosensitizer was injected intravenously to pigs at 2.5-5.0 mg/kg. About 15 min after
the injection, the laser light at the wavelength of 663 nm with a catheter output power density of 40-60 W/cm2 in about
1.4 mm spot size was irradiated through the laser catheter point by point in line crossing the isthmus under the
fluoroscopic guidance. Before the photosensitization procedure, pacing signal from the distal electrodes of the diagnostic
catheter, propagated through the isthmus in order. During the irradiation, electrical potential at the irradiated area was
diminished. After the completion of the irradiation line, the bidirectional conduction block on the IVC-TA isthmus was
validated by pacing from the distal and proximal bipole. These results indicated that photosensitization reaction could
achieve the electrical conduction block of myocardial tissue immediately after the irradiation. We think that
photosensitization reaction could become a novel therapy for atrial arrhythmia.
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.
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 propose the application of early state photodynamic therapy (PDT) to treatment of atrial fibrillation, which is a kind
of arrhythmia characterized by irregular rapid beating of heart. We had demonstrated that our PDT can block the propagation of electrical excitation in cardiac myocytes. However, the mechanism of the PDT-induced electrical blockade was not clear. In order to clarify this mechanism, changes in intracellular Ca2+ concentration during the PDT with Talaporfin sodium (water soluble photosensitizer) were measured by fluorescence Ca2+ indicator, fluo-4 AM. The PDT led to the rapid increase of intracellular Ca2+ concentration and the changes in cell shapes. These results indicated that extracellular Ca2+ flowed into the cells mediated by cell membrane. Moreover, we found bubble generation in the cells after the PDT. In conclusion, the PDT-induced electrical blockade in myocytes can be caused by cell death following the bubble generation, which is accompanied by the increase in intracellular Ca2+ concentration due to the cell membrane malfunction with the PDT.
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