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 photosensitization reaction progress in a cell culture well by oxygen partial pressure distribution measurement along the well depth direction with a high concentration of talaporfin sodium solution. The talaporfin sodium solution of 20 μg/ml in concentration with 2.8 mm thickness in the well was irradiated from the well bottom by 663 nm excitation laser with 0.29 W/cm2. A small Clark-type oxygen electrode was used to measure oxygen partial pressure during the photosensitization reaction with approximately 200 μm resolution. Corrections against solution temperature change and direct light irradiation were applied to the electrode output. The oxygen partial pressures at various depths were decreased uniformly from the atmospheric oxygen partial pressure with the photosensitization reaction progress up to the irradiation of 1.0 J/cm2 in radiant exposure. In the case of photosensitization reaction over 1.0 J/cm2 in radiant exposure, the oxygen partial pressure distribution along the well depth was non-uniform. In the case of photosensitization reaction with 40 J/cm2 in radiant exposure in the solution without cells, there was pressure gradient of 2.8×104 mmHg/m from 0.5 to 1.0 mm in depth from the solution surface. In this case, there was no pressure gradient near the bottom of the well. In contrast, with myocardial cells at the bottom, oxygen partial pressure gradient of 7.5×103 mmHg/m from 1.5 to 2.0 mm in the depth was appeared after irradiation with 40 J/cm2 in radiant exposure. Consequently, we found that oxygen partial pressure distribution along the depth in the well with high concentration of talaporfin sodium solution was dynamically changed with time of the photosensitization reaction using the laser irradiation from the bottom. We think this dynamic pressure change in the well might be useful to understand the photosensitization reaction progress in the well experiment system in vitro corresponding to the extracellular PDT.
In order to understand extracellular-photosensitization reaction (PR) using talaporfin sodium, we studied comparison of oxidation dynamics of albumin and talaporfin sodium in solution system by visible and ultraviolet absorption spectrum measurements. Almost all talaporfin sodium particles may be bound to albumin in interstitial fluid, and this binding would affect the oxidation dynamics during this PR. Bovine serum albumin (BSA) is commonly used in vitro study but its binding characteristics with talaporfin sodium are different from human serum albumin (HSA). PR was operated in a solution composed of 20 μg/ml talaporfin sodium and 1.3 mg/ml HSA or BSA to simulate myocardial extracellular PR condition. Laser radiation of 662 nm was irradiated to this solution with irradiance of 0.29 W/cm2. Absorption spectra of these solutions were measured during the PR. We estimated oxidized ratio by absorption difference around 240 nm before and after the PR. Talaporfin sodium was oxidized 100% with HSA and BSA by the PR of 100 J/cm2 in radiant exposure. On the other hand, HSA and BSA were oxidized 60% and 94%, respectively in this radiant exposure. Q-band absorption peak of talaporfin sodium with HSA was shifted to 1 nm longer wavelength increasing radiant exposure up to 100 J/cm2. This longer wavelength shift would mean binding ratio of non-oxidized talaporfin sodium to non-oxidized HSA was increased with increasing radiant exposure. Therefore it would be possible that PR with talaporfin sodium bound to HSA might present efficient PDT than PR bound to BSA.
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
To quantitatively investigate photosensitization reaction in vitro against myocardial cells with photosensitizer rich
condition in solution using Talaporfin sodium in the well of a 96 well plate, we studied photosensitization reaction
progress in this well. We have proposed non-thermal conduction block of myocardium tissue using the photosensitization reaction with laser irradiation shortly after Talaporfin sodium injection. In above situation, the
photosensitizer is located outside the myocardial cells in high concentration. To understand interaction of the
photosensitization reaction in which the photosensitizer distributes outside cells, the photosensitization reaction progress in the well was studied. Talaporfin sodium (799.69 MW) solution and a 663 nm diode laser were used. The
photosensitizer solution concentrations of 12.5-37.5 μM were employed. The photosensitizer fluorescence with 0.29
W/cm2 in irradiance, which was optimized in previous cell death study, was measured during the laser irradiation until 40 J/cm2. The photosensitizer solution absorbance and dissolved oxygen pressure after the laser irradiation were also measured. We found that the photosensitization reaction progress had 2 distinctive phases of different reaction rate: rapid photosensitization reaction consuming dissolved oxygen and gentle photosensitization reaction with oxygen diffusion from the solution-air boundary. The dissolved oxygen pressure and photosensitizer solution absorbance were 30% and 80% of the initial values after the laser irradiation, respectively. Therefore, oxygen was rate-controlling factor of the photosensitization reaction in the well with the photosensitizer rich condition. In the oxygen diffusion phase, the oxygen pressure was maintained around 40 mmHg until the laser irradiation of 40 J/cm2 and it is similar to that of myocardium tissue in vivo. We think that our 96 well plate in vitro system may simulate PDT in myocardial tissue with photosensitization reaction parameters mentioned above.
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 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.
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